AU2020217894B2

AU2020217894B2 - Polynucleotides

Info

Publication number: AU2020217894B2
Application number: AU2020217894A
Authority: AU
Inventors: Romuald Corbau; Azadeh Kia; Jenny MCINTOSH; Carlos Miranda; Amit Nathwani
Original assignee: Spur Therapeutics Ltd
Current assignee: Spur Therapeutics Ltd
Priority date: 2019-02-04
Filing date: 2020-02-04
Publication date: 2025-08-21
Anticipated expiration: 2040-02-04
Also published as: JP2022520179A; JP7600124B2; CN120758533A; SG11202108502UA; EP3850089B1; EP3850089A1; BR112021015312A2; KR20210148101A; IL285350B1; MX2021009305A; CA3128875A1; CN113795575A; HUE065347T2; EP4299116A2; IL285350A; HRP20240184T1; WO2020161483A1; AU2020217894A1; DK3850089T5; US12331331B2

Abstract

The present invention relates to polynucleotides comprising a GBA nucleotide sequence that encodes a GCase protein or fragment thereof and wherein a portion of the coding sequence is not wild type. The present invention further relates to viral particles comprising a recombinant genome comprising the polynucleotide of the invention, compositions comprising the polynucleotides or viral particles, and methods and uses of the polynucleotides, viral particles or compositions.

Description

POLYNUCLEOTIDES

Field of the Invention

The present invention relates to polynucleotides comprising a GBA nucleotide sequence

encoding B-Glucocerebrosidase ß-Glucocerebrosidase (GCase), viral particles comprising the polynucleotides

and treatments utilising the polynucleotides.

Background to the Invention

Gaucher disease (GD) is an autosomal recessive lipid storage disease characterised by the

deposition of glucocerebroside in cells of the macrophage-monocyte system. GD is caused

by mutations in the housekeeping GBA gene that impairs activity and/or production of the

enzyme B-Glucocerebrosidase ß-Glucocerebrosidase (GCase).

There are three major types of GD which are characterised by the specific mutations which

have been identified, and each type can display differing clinical symptoms. Type 1 GD

has little or no involvement with the central nervous system but is mainly characterised by

visceral manifestations such as enlarged spleen and liver, low blood cell counts, bleeding

problems and bone disease. For the past 20 years, enzyme replacement therapy has

emerged as the standard of care for type 1 GD. In addition to its high cost (~$200,000 or

~£150,000/patient/year), enzyme replacement therapy treatment in GD generally requires

one or more injections every other week for life. This leads to a high proportion of GD

patients displaying high levels of treatment burden.

Accordingly, there is a need to provide an effective therapy vector for the treatment of GD,

i.e. one that allows for a high level of GCase expression.

The present application relates to a gene therapy approach for treating GD, involving

administering a viral particle comprising a GBA polynucleotide encoding GCase. The

polynucleotides and viral particles described herein can provide higher GCase expression compared to polynucleotides comprising a wild type GCase encoding polynucleotides. 24 Jul 2025

Such a gene therapy approach would avoid the need for frequent and lifelong intravenous injections of GCase.

5 Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of the common general 2020217894

knowledge in the field. Summary of the Invention

10 In a first form, the present invention provides a polynucleotide comprising a GBA nucleotide sequence, wherein the GBA nucleotide sequence encodes a β-Glucocerebrosidase (GCase) protein and wherein the GBA nucleotide sequence comprises a sequence that is: (i) 100% identical to SEQ ID NO: 1 or SEQ ID NO: 5; (ii) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at 15 least 99.8% identical to SEQ ID NO: 1 or SEQ ID NO: 5, wherein the GCase encoded by the GBA nucleotide sequence has GCase activity and expresses in human liver cells at higher levels compared to a GCase encoded by a wild type GBA nucleotide sequence in an otherwise identical reference polynucleotide; and/or (iii) a variant of SEQ ID NO: 1 or SEQ ID NO: 5 encoding a GCase protein having GCase 20 activity, wherein the variant is identical to SEQ ID NO: 1 or SEQ ID NO: 5 respectively except that it comprises nucleotide substitutions such that the GCase protein has 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, or up to 10 amino acid substitutions relative to the wild type GCase amino acid sequence of SEQ ID NO: 25, further wherein the GCase encoded by the GBA nucleotide expresses in human liver cells at higher levels 25 compared to a GCase encoded by a wild type GBA nucleotide sequence in an otherwise identical reference polynucleotide.

In a second form, the present invention provides a viral particle comprising a recombinant genome comprising the polynucleotide of the first form. 30

In a third form, the present invention provides a composition comprising the 24 Jul 2025

polynucleotide of the first form or the viral particle of the second form; and a pharmaceutically acceptable excipient.

5 In a fourth form, the present invention provides a method of treatment comprising administering an effective amount of the polynucleotide of the first form, the viral particle of 2020217894

the second form or the composition of the third form, wherein the method is (a) a method of treating Gaucher disease; (b) a method of treating Gaucher disease wherein the Gaucher disease is type I, II or III; 10 and/or (c) a method of treating Gaucher disease wherein the patient has antibodies or inhibitors to a recombinant GCase with which the patient has previously been treated as part of an enzyme replacement therapy.

15 In a fifth form, the present invention provides a method of expressing a GBA nucleotide sequence and (i) achieving a stable GCase activity in a subject and/or (ii) providing greater GCase bioavailability in a subject compared to the bioavailability from GCase enzyme replacement therapy, wherein the bioavailability is measured over a period of 2 weeks from administration, the method comprising administering to the subject an effective amount of 20 the polynucleotide of the first form, the viral particle of the second form or the composition of the third form.

In a sixth form, the present invention provides a method of reducing hexosylceramide and/or hexosylsphingosine levels in a subject suffering from a disease or condition associated 25 with GCase deficiency, (a) the method comprising administering to the subject an effective amount of the polynucleotide of the first form, the viral particle of the second form or the composition of the third form; or (b) the method comprising administering to the subject an effective amount of the 30 polynucleotide of the first form, the viral particle of the second form or the composition of the third form, wherein reducing hexosylceramide and/or hexosylsphingosine levels leads to the treatment of the disease or condition associated with GCase deficiency.

2a

In a seventh form, the present invention provides use of an effective amount of the 24 Jul 2025

polynucleotide of the first form, the viral particle of the second form or the composition of the third form in the manufacture of a medicament for treatment of: (i) Gaucher disease; 5 (ii) Gaucher disease wherein the Gaucher disease is type I, II or III; and/or (iii) Gaucher disease wherein the patient has antibodies or inhibitors to a recombinant GCase with which the patient has previously been treated as part of an enzyme replacement therapy. 2020217894

In an eighth form, the present invention provides use of an effective amount of the 10 polynucleotide of the first form, the viral particle of the second form or the composition of the third form in the manufacture of a medicament for reducing hexosylceramide and/or hexosylsphingosine levels in a subject suffering from a disease or condition associated with GCase deficiency, wherein reducing hexosylceramide and/or hexosylsphingosine levels leads to the treatment of the disease or condition associated with GCase deficiency. 15 The present application demonstrates that specific modifications to a GBA nucleotide sequence encoding for GCase can help to improve the expression level and the activity of the expressed GCase polypeptide in vitro and/or in vivo. For example, the present application demonstrates that using a codon-optimised GBA nucleotide sequence can improve the expression and/or activity of the encoded GCase protein. Such modified 20 (i.e. non wild-type) and/or codon-optimised GBA nucleotide sequences may be further modified to provide further improvements in the expression and/or activity of the encoded GCase protein. Further modifications may include providing further modifications in the GBA nucleotide sequence such as the removal of CpG motifs, and/or the use of particular gene regulatory elements comprising specific promoter 25 and/or enhancer sequences. It is believed that such improvements to a GBA nucleotide sequence can improve the efficacy of such a nucleotide in the treatment of GD.

These modifications provide a GBA nucleotide sequence which is expressed highly, for example in the liver, and which encodes a GCase polypeptide or fragment thereof. As 30 demonstrated in the Examples, the polynucleotides of the invention express Gcase activity at higher levels than wild type GBA.

Accordingly, in a first aspect of the invention, there is provided a polynucleotide

2b comprising a GBA nucleotide sequence, wherein the GBA nucleotide sequence encodes 24 Jul 2025 a β-Glucocerebrosidase (GCase) protein or fragment thereof and wherein at least a portion of the GBA nucleotide sequence is not wild type.

5 In a second aspect of the invention, there is provided a polynucleotide comprising a GBA nucleotide sequence, wherein the GBA nucleotide sequence encodes a GCase 2020217894

protein or a

2c fragment thereof and comprises a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to a fragment of at least 1000, at least 1200, at least 1300, less than 1494, less than 1611, between 1000 and 1494, between 1000 and 1611, between 1300 and 1494, between 1300 and 1611, or around 1494 nucleotides of SEQ ID NO: 1-8.

In a third aspect of the invention, there is provided a viral particle comprising a

recombinant genome comprising the polynucleotide of the invention.

In a fourth aspect of the invention, there is provided a composition comprising the

polynucleotide or viral particle of the invention and a pharmaceutically acceptable

excipient.

In a fifth aspect of the invention, there is provided a method of treatment comprising

administering an effective amount of the polynucleotide or viral particle of the invention to

a patient.

In In a a sixth sixth aspect aspect of of the the invention, invention, there there is is provided provided a a use use of of the the polynucleotide, polynucleotide, viral viral

particle or composition of the invention in the manufacture of a medicament for use in a

method of treatment.

In a seventh aspect of the invention, there is provided the use of the polynucleotide, viral

particle or composition of the invention in the manufacture of a medicament for achieving

a stable GCase activity in a subject.

In an eighth aspect of the invention, there is provided the use of the polynucleotide, viral

particle or composition of the invention in the manufacture of a medicament for providing

greater GCase bioavailability in a subject compared to the bioavailability from GCase

enzyme replacement therapy, wherein the bioavailability is measured over a period of 2

weeks from administration.

PCT/GB2020/050251

In a ninth aspect of the invention, there is provided a method of achieving a stable GCase

activity in a subject by administering to the subject the polynucleotide, viral particle or

composition of the invention.

In a tenth aspect of the invention, there is provided a method of providing greater GCase

bioavailability in a subject compared to the bioavailability from GCase enzyme

replacement replacement therapy therapy by by administering administering to to the the subject subject the the polynucleotide, polynucleotide, viral viral particle particle or or

composition of the invention, wherein the bioavailability is measured over a period of 2

weeks from administration.

In an eleventh aspect of the invention, there is provided a polynucleotide, viral particle or

composition of the invention, for use in a method of expressing the GBA nucleotide

sequence and achieving a stable GCase activity in a subject.

In a twelfth aspect of the invention, there is provided a polynucleotide, viral particle or

sequence and providing greater GCase bioavailability in a subject compared to the

bioavailability from GCase enzyme replacement therapy, wherein the bioavailability is

measured over a period of 2 weeks from administration.

In a thirteenth aspect of the invention, there is provided the use of the polynucleotide, viral

particle or composition of the invention in the manufacture of a medicament for reducing

the levels of hexosylceramide and/or hexosylsphingosine in a subject suffering from a

disease or condition associated with GCase deficiency.

In a fourteenth aspect of the invention, there is provided a method of reducing the levels of

hexosylceramide and/or hexosylsphingosine in a subject suffering from a disease or

condition associated with GCase deficiency by administering to the subject the

polynucleotide, viral particle or composition of the invention.

WO wo 2020/161483 PCT/GB2020/050251 PCT/GB2020/050251

In a fifteenth aspect of the invention, there is provided a polynucleotide, viral particle or

composition of the invention, for use in a method of reducing levels of hexosylceramide

and/or and/or hexosylsphingosine hexosylsphingosinein ain subject suffering a subject from a disease suffering from a or condition disease associated associated or condition

with GCase deficiency, optionally wherein reducing hexosylceramide and/or

hexosylsphingosine levels leads to the treatment of the disease or condition associated with

GCase deficiency.

Description of the Figures

Figure 1 - Schematics of the GBA cassettes from the constructs FLF-PL01, FLF-PL28,

and FLF-PL64. LSP-S and LSP-L: liver specific promoters; GBAwt: wild type human

GBA nucleotide sequence; GBAco: human GBA nucleotide sequence codon-optimised

(except for stretch encoding signal peptide, the end of which is represented by a dotted

line).

Figure 2 - Dose-dependent liver expression and secretion of human GCase into murine

bloodstream following AAV2/8-FLF-PL28 injection. (A) Representative image of mouse

livers stained for GCase 12-weeks post AAV2/8-PL28 injection. DAB (3,3'-

Diaminobenzidine) was used to visualize GCase and haematoxylin was used as

counterstain. (B) Levels of GCase as measured by activity assay in serum of mice treated

with increased doses of AAV2/8-PL28. n=5, C57BL/6 mice in each treatment group. Error

bars show mean + ± SD.

Figure 3 - Relative GCase levels observed for each tested GBA codon-optimised construct

(FLF-PL16 to FLF-PL36; '16' to '36') upon transfection onto Huh-7 cells. Each construct

was tested independently in 3 to 5 experiments. Data shown here represent GCase activity

relative to wild type GBA construct FLF-PL01 ('01'). Error bars represent mean + ± SD.

Figure 4 - Measurement of GCase activity present in the mouse bloodstream upon

injection of vectors AAV2/8- FLF-PL- 01, 21, 28, 30, 36 and 37 (see example 5 for

description of constructs). (A) GCase activity levels found in mouse serum 8-weeks after

WO wo 2020/161483 PCT/GB2020/050251

injection of tested GBA constructs. (B) GCase activity levels observed in mouse serum at

4-, 8-, 12-, and 36-weeks post-injection of constructs FLF-PL01 and FLF-PL28. Error bars

represent mean + ± SD, n = 5-8 animals per experimental group. * p 0.05; 0.05;p ** 0.001 p 0.001

(one-way ANOVA).

Figure 5 - Uptake levels of GCase in spleen and bone marrow tissue following AAV2/8-

FLF-PL28 injection in wild type mice. Representative image of spleen and bone marrow

tissue stained for GBA is shown for naive naïve or AAV2/8-PL28 treatment mice at 4-weeks

post-injection. DAB (3,3'-Diaminobenzidine) post-injection. DAB was used to was visualize used to visualize GBAGBAand and

haematoxylin was used as counterstain.

Figure 6 - Levels of co-localization of human GCase with canonical murine macrophage

marker F4/80 observed in spleen upon injection of wild type mice with AAV2/8-FLF-

PL28. Representative immunofluorescence image of spleen tissue stained for GBA and

F4/80 antibodies. DAPI (Blue) was used to visualise nuclei.

Figure 7 - Levels of GCase activity found in mouse bloodstream 4 weeks post-injection of

AAV2/8-FLF-PL28 AAV2/8- FLF-PL28and andFLF-PL64. FLF-PL64.GCase GCaseactivity activitywas wasdetermined determinedfor formouse mouseserum serum collected 4-weeks post-injection at the dose of 2x1012 2x10¹² vg/kg. Error bars represents mean + ±

SD. N = 5, C57BL/6 mice in each treatment group.

Figure 8 - Uptake levels observed in spleen, bone marrow and lung following AAV2/8-

FLF-PL28 and FLF-PL64 injection observed in mice 5-weeks post treatment.

Figure 9 - Sequence listing.

Figure 10 - Levels of GCase secretion by human-derived cell lines following transduction

with AAV-FLF-PL64. Cells were transduced at a MOI of 1x105 vg/cellwith 1x10 vg/cell withthe thevector vector

AAV-FLF-PL64. (A) Levels of active GBA were determined fluorometrically with 4MU-

Glc as the substrate. (B) The level of transduction for each cell line was obtained by qPCR

using primersspecific using primers specific for for the the polyApolyA sequence. sequence. Blank for Blank values values each for celleach line cell were line were

WO wo 2020/161483 PCT/GB2020/050251 PCT/GB2020/050251

subtracted to obtain a value for the level of active GCase. Error bars represent mean 1 ± SD

of duplicate wells.

Figure 11: (A) Enzyme replacement therapy (VPRIV R (60 U/kg)) pharmacokinetics and

half-life calculation in wild type mice. One-phase decay model equation: Y0 is the Y-value

when X (time) is zero. Plateau is the Y-value at infinite times. K is the rate constant. Tau is

the time constant. Half-life is in the time units of the X-axis. Span is the difference

between Y0 and Plateau. (B) Comparison between serum pharmacokinetic profile of

GCase activity after a single injection of enzyme replacement therapy (VPRIV (60 U/kg),

solid black) and gene therapy with FLF-PL64, following administration in wild type mice.

Figure 12: GCase immunostaining in murine liver, spleen and bone following

administration of VPRIV or FLF-PL64. DAB (3,3'-Diaminobenzidine) was used to

visualise GCase and haematoxylin was used as counterstain. FLF-PL64 samples were

obtained at five weeks post-injection, while VPRIV treated samples were collected at the

time labelled. Each image represents n=5, C57BL/6 mice for each treatment group. All

pictures are at the same magnification.

Figure Figure13: 13:Increase in in Increase GCase activity GCase observed activity in gba9v/null observed mouse liver in gbav/ull mouse(a), white liver blood (a), white blood

cells (b), spleen (c), and bone marrow (d) upon administration of velaglucerase alfa

(VPRIV R) (labelled ERT) or AAV-GBA (AAV-FLF-PL64). ERT samples were

collected 1-2 hours post the last injection corresponding to peak of uptake in tissues. AAV-

GBA (AAV-FLF-PL64) samples were collected 12 weeks post-injection and

corresponding to steady state levels of uptake. GCase activity is represented as a

percentage of the activity measured in wild-type healthy mice (at 20 weeks of age). All

mice were treated at age of 8-week pre-overt symptomatology. ERT at dose 60 U/kg and

administered by injection every two weeks; AAV-FLF-PL64 injected at dose of 2x1012 2x10¹²

vg/kg. n=10. vg/kg. **** P <0.0001 n=10.****P<0.0001

Figure 14: AAV-GBA (AAV-FLF-PL64) gene therapy reduces activated macrophages and and inflammation inflammationin in thethe liver of gba9v/null liver mice. mice. of gbav/null Upper panel: H&E stained Upper panel: H&E liver sections stained liver sections showing a representative image from each group. Storage cells are identified by circles.

Lower left panel: graph showing comparison between storage cells counted in AAV-FLF-

PL64 and ERT treated groups compared to vehicle control groups. Lower right panel:

graph showing CD68positive cells counted in AAV-FLF-PL64 and ERT treated groups

compared to vehicle control groups following staining with anti-CD68 antibody. AAV-

GBA (AAV-FLF-PL64) injected at dose of 2x 1012 10¹² vg/kg; vg/kg; samples samples collected collected 12-weeks 12-weeks p.i. p.i.

ERT at dose 60 U/kg and administered by injection every two weeks. ERT samples were

collected 1-2 hours post the last injection. Mean 1 ± SEM, (n=10),** P<0.005,****PS (n=10), P0.005 , **** P

0.0005

Figure 15: AAV-GBA (AAV-FLF-PL64) gene therapy shows better substrate clearance

than velaglucerase alfa (VPRIV R, labelled ERT) in gba9v/null mice. LC/MS gbav/null mice. LC/MS analysis analysis of of

hexosylceramide hexosylceramide and and hexosylsphingosine hexosylsphingosine levels levels in in liver, liver, spleen, spleen, and and bone bone marrow marrow in in

AAV-FLF-PL64 and ERT treated groups. Levels were standardised to the levels measured

in the vehicle control group. AAV-GBA (AAV-FLF-PL64) injected at dose of 2x 1012 2x10¹²

vg/kg; samples collected 12-weeks p.i.; ERT at dose 60 U/kg and administered by injection

every two weeks. ERT samples were collected 1-2 hours post the last injection. Mean

+SEM, ±SEM, (n=10),**P<0.005,****P<0.0005 (n=10),' P0.005, **** 0.0005

Detailed Description

General definitions

Unless defined otherwise, technical and scientific terms used herein have the same

meaning as commonly understood by a person skilled in the art to which this invention

belongs.

In general, the term "comprising" is intended to mean including but not limited to. For

example, the phrase "a polynucleotide polymicleotide comprising a GBA nucleotide sequence" should be

interpreted to mean that the polynucleotide has a GBA nucleotide sequence, but the

polynucleotide may contain additional nucleotides.

WO wo 2020/161483 PCT/GB2020/050251 PCT/GB2020/050251

In some embodiments of the invention, the word "comprising" is replaced with the phrase

"consisting of" of' The term "consisting of of'is isintended intendedto tobe belimiting. limiting.For Forexample, example,the the

phrase "a polymucleotide polymicleotide consisting of a GBA nucleotide sequence" should be understood

to mean that the polynucleotide has a GBA nucleotide sequence and no additional

nucleotides. nucleotides.

As used herein, "between" when referring to two endpoints to define a range of values

should be taken to mean "between and including". Thus, a range defined as "between 5

and 10" includes all values greater than 5 and less than 10, as well as the discrete values 5

and 10 themselves.

The terms "protein" and "polypeptide" are used interchangeably herein, and are intended

to refer to a polymeric chain of amino acids of any length.

For the purpose of this invention, in order to determine the percent identity of two

sequences (such as two polynucleotide or two polypeptide sequences), the sequences are

aligned for optimal comparison purposes (e.g., gaps can be introduced in a first sequence

for optimal alignment with a second sequence). The nucleotide or amino acid residues at

each position are then compared. When a position in the first sequence is occupied by the

same nucleotide or amino acid residue as the corresponding position in the second

sequence, then the nucleotides or amino acids are identical at that position. The percent

identity between the two sequences is a function of the number of identical positions

shared by the sequences (i.e., % identity = number of identical positions /total number of

positions in the reference sequence X 100).

Typically the sequence comparison is carried out over the length of the reference sequence.

For example, if the user wished to determine whether a given ("test") sequence is 95%

identical to SEQ ID NO: 1, SEQ ID NO: 1 would be the reference sequence. For example,

to assess whether a sequence is at least 80% identical to SEQ ID NO: 1 (an example of a

reference sequence), the skilled person would carry out an alignment over the length of

SEQ ID NO: 1, and identify how many positions in the test sequence were identical to

those of SEQ ID NO: 1. If at least 80% of the positions are identical, the test sequence is

at least 80% identical to SEQ ID NO: 1. If the sequence is shorter than SEQ ID NO: 1, the

gaps or missing positions should be considered to be non-identical positions.

The skilled person is aware of different computer programs that are available to determine

the homology or identity between two sequences. For instance, a comparison of sequences

and determination of percent identity between two sequences can be accomplished using a

mathematical algorithm. In an embodiment, the percent identity between two amino acid

or nucleic acid sequences is determined using the Needleman and Wunsch (1970)

algorithm which has been incorporated into the GAP program in the Accelrys GCG

software package (available at http://www.accelrys.com/products/gcg/), using either a

Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a

length weight of 1, 2, 3, 4, 5, or 6.

For the purposes of the present invention, the term "fragment" refers to a contiguous

portion of a sequence. For example, a fragment of SEQ ID NO: 1 of 50 nucleotides refers

to 50 contiguous nucleotides of SEQ ID NO: 1.

A polynucleotide polymicleotide

In one aspect, the present invention provides a polynucleotide comprising a GBA

nucleotide sequence, wherein the GBA nucleotide sequence encodes a B- ß-

Glucocerebrosidase (GCase) protein or fragment thereof and wherein at least a portion of

the GBA nucleotide sequence is not wild type.

The polynucleotide may further comprise one or more of the following features. The GBA

nucleotide sequence, or portion of GBA nucleotide sequence that is not wild type, may be

codon-optimised. The polynucleotide may (additionally) comprise a portion that is not

codon-optimised. The polynucleotide may comprise an intron or a fragment of an intron.

WO wo 2020/161483 PCT/GB2020/050251

The term "polymucleotide" "polymicleotide" refers to a polymeric form of nucleotides of any length,

deoxyribonucleotides, ribonucleotides, or analogs thereof. For example, the

polynucleotide may comprise DNA (deoxyribonucleotides) or RNA (ribonucleotides). The

polynucleotide may consist of DNA. The polynucleotide may be mRNA. Since the

polynucleotide may comprise RNA or DNA, all references to T (thymine) nucleotides may

be replaced with U (uracil).

A GBA nucleotide sequence encoding GCase

In one aspect, the polynucleotide provided herein comprises a GBA nucleotide sequence.

The GBA nucleotide sequence typically encodes the B-Glucocerebrosidase (GCase) protein -Glucocerebrosidase (GCase) protein

or fragment thereof.

The term "sequence that encodes" refers to a nucleotide sequence comprising an open

reading frame comprising codons that encode the encoded polypeptide. For example, a

nucleotide sequence that encodes a GCase protein or fragment thereof comprises codons

that encode the amino acid sequence of a GCase protein or fragment thereof. An example

of a GBA nucleotide sequence that encodes a wild type GCase protein is provided in SEQ

ID NO: 9.

A GBA nucleotide sequence may be interrupted by non-coding nucleotides (e.g. an intron),

but only nucleotides that encode the polypeptide should be considered to be part of the

GBA nucleotide sequence. For example, a GBA nucleotide sequence that encodes a

GCase protein will comprise any codons that encode an amino acid forming part of the

GCase protein that is expressed from that coding sequence, irrespective of whether those

codons are contiguous in sequence or separated by one or more non-coding nucleotides. In

other words, a GBA polynucleotide which contains stretches of coding nucleotides

interrupted by a stretch of non-coding nucleotides will be considered to comprise a "GBA

nucleotide sequence" consisting of the non-contiguous coding stretches immediately

juxtaposed (i.e. minus the non-coding stretch). However, herein, the stop codon will be

considered to be part of the full length coding sequence.

A GBA nucleotide sequence encoding GCase and/or a GCase coding sequence as

described herein may also include codons for a signal peptide. It is well known that some

proteins, particularly those which are exported to different tissues, are expressed with a

signal peptide. Signal peptides can be at the N-terminus of a protein sequence (and in this

case at the 5' end of a coding sequence) and many signal peptides are cleaved following

cellular processing. Thus, herein, a mature protein or polypeptide (such as a mature GCase

protein or polypeptide) will be considered to be the resulting protein or polypeptide after

the signal peptide has been processed and removed/cleaved (and thus no longer forms part

of the polypeptide sequence).

The following Table describes codons that encode each amino acid:

Amino Acid Codon Amino Codon Amino Acid Codon Acid Phenylalanine Proline Asparagine TTC CCT AAT TTT TTT CCC AAC CCA CCG Leucine TTA Threonine ACT Lysine AAA TTG ACC AAG CTT ACA ACA CTC ACG ACG CTA CTG Isoleucine Alanine Aspartic Acid ATT GCT GAT ATC GCC GAC ATA GCA GCG Methionine Tyrosine Glutamic ATG TAT TAT GAA Acid TAC GAG Valine Histidine Cysteine GTT CAT TGT GTC GTC CAC TGC GTA GTG Serine Glutamine Tryptophan TCT CAA TGG TCC CAG TCA TCG AGT AGC Arginine Glycine Glycine CGT GGT

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CGC GGC CGA GGA CGG GGG AGA AGG

The corresponding RNA codons will contain Us in place of the Ts in the Table above.

One aspect of the present invention provides a polynucleotide comprising a GBA

nucleotide sequence, wherein the GBA nucleotide sequence encodes a GCase protein or a

fragment thereof and comprises a sequence that is at least 95%, at least 96%, at least 97%,

at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to a fragment

of at least 1000, at least 1200, at least 1300, less than 1494, less than 1611, between 1000

and 1494, between 1000 and 1611, between 1300 and 1494, between 1300 and 1611, or

around 1494 nucleotides of any one of SEQ ID NO: 1-8. Optionally, all or a portion of

the GBA nucleotide sequence is codon-optimised. In one embodiment, the GBA

nucleotide sequence comprises a sequence that is at least 98% identical to a fragment of at

least 1300 nucleotides of SEQ ID NO: 1-8. In one embodiment, the GBA nucleotide

sequence sequence comprises comprises aa sequence sequence that that is is at at least least 99% 99% identical identical to to aa fragment fragment of of at at least least 1300 1300

nucleotides of SEQ ID NO: 1-8.

In one example, the GBA nucleotide sequence may comprise a sequence that is at least

95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ

ID NO: 1. In one embodiment, the GBA nucleotide sequence comprises a sequence that is

at least 98% identical to a fragment of at least 1300 nucleotides of SEQ ID NO: 1. In one

embodiment, the GBA nucleotide sequence comprises a sequence that is at least 99%

identical to a fragment of at least 1300 nucleotides of SEQ ID NO: 1. The GBA

nucleotide sequence may comprise a sequence that is at least 95%, at least 98%, at least

99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 5. The GBA

nucleotide sequence may comprise a sequence that is at least 98% identical SEQ ID NO: 1.

The GBA nucleotide sequence may comprise a sequence that is at least 99% identical SEQ

ID NO: 1. The GBA nucleotide sequence may comprise a sequence that is at least 98%

identical SEQ ID NO: 5. The GBA nucleotide sequence may comprise a sequence that is

PCT/GB2020/050251

at least 99% identical SEQ ID NO: 5. In one embodiment, the GBA nucleotide sequence

may comprise SEQ ID NO: 1. In another embodiment, the GBA nucleotide sequence may

comprise SEQ ID NO: 5.

The GBA nucleotide sequence may comprise a sequence of SEQ ID NO: 1 or a variant of

SEQ ID NO: 1 encoding a GCase protein having GCase activity. In these examples, a

variant of SEQ ID NO: 1 is identical to SEQ ID NO: 1 except that it comprises nucleotide

substitutions such that the GCase protein has 1, up to 2, up to 3, up to 4, up to 5, up to 6, up

to 7, up to 8, up to 9, or up to 10 amino acid substitutions relative to the wild type GCase

amino acid sequence of SEQ ID NO: 25. In these examples, the variant of SEQ ID NO: 1

may have 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up up to 20, or up to 30 nucleotide substitutions relative to the sequence of SEQ ID NO: 1. The

variant variant of of SEQ SEQ ID ID NO: NO: 11 may may have have 1, 1, up up to to 2, 2, up up to to 3, 3, up up to to 4, 4, up up to to 5, 5, or or up up to to 66

nucleotide substitutions relative to the sequence of SEQ ID NO: 1. In one example, the

variant of SEQ ID NO: 1 has up to 4 nucleotide substitutions relative to the sequence of

SEQ ID NO: 1 and/or encodes a GCase protein having up to 3 amino acid substitutions

relative to the wild type amino acid GCase sequence of SEQ ID NO: 25. In one example,

the variant of SEQ ID NO: 1 has up to 3 nucleotide substitutions relative to the sequence of

SEQ ID NO: 1 and/or encodes a GCase protein having up to 2 amino acid substitutions

relative to the wild type GCase amino acid sequence of SEQ ID NO: 25. In one example,

the variant of SEQ ID NO: 1 has 1 nucleotide substitution relative to the sequence of SEQ

ID NO: 1 and/or encodes a GCase protein having up to 1 amino acid substitution relative to

the wild type GCase amino acid sequence of SEQ ID NO: 25.

The GBA nucleotide sequence may comprise a sequence of SEQ ID NO: 5 or a variant of

SEQ ID NO: 5 encoding a GCase protein having GCase activity. In these examples, a

variant of SEQ ID NO: 5 is identical to SEQ ID NO: 5 except that it comprises nucleotide

amino acid sequence of SEQ ID NO: 25. In these examples, the variant of SEQ ID NO: 5 5 may have 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up

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to 20, or up to 30 nucleotide substitutions relative to the sequence of SEQ ID NO: 5. The

variant variant of of SEQ SEQ ID ID NO: NO: 55 may may have have 1, 1, up up to to 2, 2, up up to to 3, 3, up up to to 4, 4, up up to to 5, 5, or or up up to to 66

nucleotide substitutions relative to the sequence of SEQ ID NO: 5. In one example, the

variant ofSEQ variant of SEQIDID NO:NO: 5 has 5 has up4 to up to 4 nucleotide nucleotide substitutions substitutions relative relative to theofsequence of to the sequence

SEQ ID NO: 5 and/or encodes a GCase protein having up to 3 amino acid substitutions

the the variant variant of of SEQ SEQ ID ID NO: NO: 55 has has up up to to 33 nucleotide nucleotide substitutions substitutions relative relative to to the the sequence sequence of of

SEQ ID NO: 5 and/or encodes a GCase protein having up to 2 amino acid substitutions

the variant of SEQ ID NO: 5 has 1 nucleotide substitution relative to the sequence of SEQ

ID NO: 5 and/or encodes a GCase protein having up to 1 amino acid substitution relative to

the wild type GCase amino acid sequence of SEQ ID NO: 25.

GCase protein or fragment thereof

The polynucleotide comprises a GBA nucleotide sequence that encodes a GCase protein or

fragment thereof.

B-glucocerebrosidase ß-glucocerebrosidase (GCase) is an enzyme with glucosylceramidase activity (EC

3.2.1.45) that hydrolyses the beta-glucosidic linkage of the chemical glucocerebroside, an

intermediate in glycolipid metabolism that is abundant in cell membranes. Mutations in

the GBA gene (which encodes GCase) can lead to an accumulation of glucocerebrosides in

macrophages that infiltrate many vital organs, which manifests as Gaucher disease (GD).

A typical wild type GCase polypeptide is encoded by SEQ ID NO: 9.

GCase (e.g. a GCase of SEQ ID NO: 25 encoded by SEQ ID NO: 9) is initially expressed

as a precursor "immature" form, comprising a signal peptide (amino acid residues 1 to 39

of SEQ ID NO: 25 and codons 1 to 39 of SEQ ID NO: 9), and a mature GCase polypeptide

region. After processing, the "mature" form of GCase lacks the signal peptide. The term

"mature GCase" or "mature GCase polypeptide" refers to a GCase polypeptide that does

not comprise the signal peptide, such as a GCase encoded by SEQ ID NOs: 1-4. A typical

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GCase signal peptide may be encoded by a nucleotide sequence of SEQ ID NO: 17 and

have the polypeptide sequence of SEQ ID NO: 18.

The GCase or fragment thereof may be a variant GCase or fragment thereof, i.e. a GCase

that does not have a sequence identical to SEQ ID NO: 25. In an embodiment, the GCase

or fragment thereof that is encoded by a polypeptide of the present invention and/or by a

GBA nucleotide sequence is at least 95%, at least 98%, at least 99%, at least 99.5%, at

least 99.8%, or 100% identical to SEQ ID NO: 25; or at least 95%, at least 98%, at least

99%, at least 99.5%, at least 99.8%, or 100% identical to a fragment of SEQ ID NO: 25 at

least 300, at least 350, at least 400, less than or equal to 536, less than or equal to 497,

between 300 and 536, or between 300 and 497 amino acids in length. In an embodiment,

the GCase protein or fragment thereof is at least 95%, at least 98%, at least 99%, at least

99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 25; or at least 95%, at least 98%,

at least 99%, at least 99.5%, at least 99.8%, or 100% identical to a fragment of SEQ ID

NO: 25 about 497 amino acids in length. The GCase protein or fragment thereof may have

a sequence of SEQ ID NO: 25. Preferably the GCase protein or a fragment thereof does

not comprise the signal peptide of SEQ ID NO: 18. Preferably the GCase protein or

fragment thereof is functional. A functional GCase protein or fragment is one which

carries out hydrolysis of glucocerebroside.

The GBA nucleotide sequence may encode a GCase protein having 1, up to 2, up to 3, up

to 4, or up to 5 amino acid substitutions relative to the wild type GCase amino acid

sequence of SEQ ID NO: 25. In such examples, the GBA nucleotide sequence may encode

a GCase protein having up to 3 amino acid substitutions relative to the wild type GCase

amino acid sequence of SEQ ID NO: 25. The GBA nucleotide sequence may encode a

GCase protein having up to 2 amino acid substitutions relative to the wild type GCase

amino acid sequence of SEQ ID NO: 25. The GBA nucleotide sequence may encode a

variant GCase protein having up to 1 amino acid substitution relative to the wild type

GCase amino acid sequence of SEQ ID NO: 25.

16

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It is within the abilities of the person skilled in the art to determine whether a GCase

protein or fragment encoded by a GBA nucleotide sequence is functional. The skilled

person merely needs to express the GCase nucleotide sequence, and test whether the

expressed protein is active. For example, the skilled person could prepare a viral particle

of the invention comprising a GBA nucleotide sequence linked to an operable promoter,

and transduce cells with the viral particle under conditions suitable for expression of the

GCase protein or fragment thereof. The activity (amount) of the expressed GCase protein

or fragment thereof can be analysed using a a fluorometric, assay, such as the "serum GBA

activity assay" described in Example 1.

For example, a suitable fluorogenic assay is as follows. B-Glucocerebrosidase (acid ß- -Glucocerebrosidase (acid B-

glucosidase; GCase) activity can be determined fluorometrically with 4-

Methylumbelliferyl-B-D-glucopyranoside (4MU-Glc) as a substrate. Briefly, serum Methylumbelliferyl-ß-D-glucopyranoside

samples (0.5 uL, µL, diluted 1:50) can be assayed in 50 mM Sodium Citrate, 25 mM

Taurocholate, pH ~5.75, 6 mM 4MU-Glc, for 30 min at 37°C. Relative fluorescence levels

(RFU) may then be evaluated using excitation and emission wavelengths of 365 nm and

445 nm, respectively. GCase is expressed as nanomoles/h/ mL of serum based on a 4-

Methylumbelliferone (4-MU) standard curve.

A portion of the GBA nucleotide sequence is not wild type

A portion of the GBA nucleotide sequence, for example the coding sequence that encodes

GCase protein or a fragment thereof, may not be wild type. The wild type GCase-

encoding GBA nucleotide sequence is represented by SEQ ID NO: 9, and a GBA

nucleotide sequence that comprises a portion differing in sequence from that of SEQ ID

NO: 9 comprises a portion that is not wild type.

In an embodiment, the portion of the GBA nucleotide sequence that is not wild type is

codon-optimised. Codon-optimisation can improve expression of the nucleotide sequence,

for example a GBA nucleotide sequence, in a particular tissue and/or in a particular

organism. For example, if a nucleotide sequence is codon-optimised for expression in the

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human liver, the nucleotide sequence is modified to increase the number of codons that

may be favoured (in the sense that such codons correspond to tRNA species which are

more abundant than other tRNA species specific for the same amino acid) in the human

liver. The skilled person would appreciate that codon-optimising a sequence may not

entail changing every codon, not least because a "favoured codon" may already be present

at some positions.

Such codon-optimisation may be subject to other factors. For example, it can be seen that

the presence of CpGs has an adverse effect on expression and SO so the user may decide not to

use favoured codons at positions where doing SO so introduces CpGs into the sequence; this

will still be considered to be codon-optimisation. In an embodiment, a favoured codon that

ends with a C nucleotide will not be included in the portion of the coding sequence that is

codon-optimised, where the next codon in the sequence begins with a G. For example,

codon CTC encodes leucine. In schemes where CTC is a favoured codon, it should not be

used for encoding leucine where the next codon in the sequence begins with a G, such as

codon GTT (or alternatively, the next codon - where possible - could be selected to avoid

a G at the first position).

It is straightforward to determine the frequency of each codon used in a portion of a

nucleotide sequence. The skilled person merely needs to enter the sequence of that portion

into one of the readily-available algorithms that looks at codon usage and review the

results. Alternatively, the user could simply count them.

In one embodiment, a polynucleotide of the invention comprises a GBA nucleotide

sequence wherein 67% of the codons that encode histidine are CAC and 33% of the codons

that encode histidine are CAT.

Optionally, the portion of the GBA nucleotide sequence that is codon-optimised is codon-

optimised for expression in human liver cells. Optionally, the GBA nucleotide sequence is

codon-optimised for expression in the human liver. Optionally, the portion of the GBA

nucleotide sequence that is codon-optimised is a contiguous portion.

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The portion that is codon-optimised can correspond to a sequence encoding part of, or an

entire, GCase protein. For example, the coding sequence could be full-length (such as

SEQ ID NO: 9), including the signal peptide which is not part of the mature GCase protein,

and the entire coding sequence could be codon-optimised. Hence, reference herein to "a

portion of the GBA sequence is codon-optimised" should be understood to mean "at least

a portion of the GBA sequence is codon-optimised". Optionally, the portion of the GBA

nucleotide sequence that is codon-optimised is at least 1000, at least 1200, at least 1300,

less than 1600, less than 1500, between 1000 and 1600, between 1000 and 1500, between

1300 and 1500, or around 1494 nucleotides in length. Optionally, the portion of the GBA

nucleotide sequence that is codon-optimised encodes (corresponds to) a mature GCase

protein. For example, the GBA nucleotide sequence may encode a precursor GCase protein

(i.e. (i.e. including including signal signal peptide), peptide), and and if if the the portion portion of of the the GBA GBA nucleotide nucleotide sequence sequence that that is is

codon-optimised corresponds to the mature GCase protein, the signal peptide is not codon-

optimised.

Thus in some embodiments, a portion of the GBA nucleotide sequence may not be codon-

optimised, for example a portion of the coding sequence is not codon-optimised for

expression in the liver. In some embodiments, the portion that is not codon-optimised is at

least 80, at least 90, at least 100, at least 110, less than 200, less than 170, less than 140, or

around 117 nucleotides. In some embodiments, the portion that is not codon-optimised in

a GBA nucleotide sequence is the portion which encodes the signal peptide.

As discussed above, providing a polynucleotide sequence comprising a GBA nucleotide

sequence that is partially or wholly codon-optimised can ensure that the encoded

polypeptide (i.e. a GCase polypeptide) is expressed at a high level. It will be appreciated

by one skilled in the art that expression of GCase from a polynucleotide sequence, such as

a GBA nucleotide sequence of the present invention, or from a viral particle of the present

invention, generally requires the presence of a promoter sequence or region upstream of

and/or operably linked to the polynucleotide sequence. Thus in one embodiment, the

present invention provides a polynucleotide comprising a GBA nucleotide sequence,

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wherein the GBA nucleotide sequence encodes a GCase polypeptide which is expressed in

human liver cells at high levels when the GBA nucleotide sequence is operably linked to a

promoter sequence. In some embodiments, the promoter sequence may be part of a

transcriptional transcriptional regulatory regulatory element. element. In In some some embodiments, embodiments, the the promoter promoter sequence sequence may may be be aa

liver-specific promoter sequence. In one embodiment, the promoter sequence is a

promoter having SEQ ID NO: 12. In another embodiment, the promoter sequence is a

promoter havingSEQ promoter having SEQ ID ID NO:NO: 15. 15.

It will also be appreciated by one skilled in the art that making comparisons between

polynucleotides or vectors of the invention and reference (comparator) polynucleotides or

vectors such as a reference polynucleotide or a viral particle comprising a GBA nucleotide

sequence of SEQ ID NO: 9, the reference polynucleotides or vectors may be identical to

the polynucleotides or vectors of the invention except that the GBA nucleotide sequences

are different. In other words, the different GBA nucleotide sequences being compared

may be operably linked to the same promoter sequence. In some embodiments, the

different different GBA GBA nucleotide nucleotide sequences sequences being being tested tested may may be be operably operably linked linked to to different different

(specified) promoter sequences.

Thus, in one embodiment, a GCase polypeptide encoded by the GBA nucleotide sequence

is expressed in human liver cells at higher levels compared to a reference wild type GBA

sequence. The reference wild type GBA nucleotide sequence may be SEQ ID NO: 9. In

an embodiment, a polypeptide encoded by the GBA nucleotide sequence is expressed in

human liver cells at higher levels compared to a polypeptide encoded by a nucleotide

sequence comprising a GBA nucleotide sequence of SEQ ID NO: 9 and a promoter

element of SEQ ID NO: 13 (wherein the GBA nucleotide sequence of SEQ ID NO: 9 and

the the promoter promoter element element of of SEQ SEQ ID ID NO: NO: 13 13 are are preferably preferably operably operably linked). linked). In In an an

embodiment, a polypeptide encoded by the GBA nucleotide sequence is expressed in

sequence comprising a GBA nucleotide sequence of SEQ ID NO: 9 and a transcription

regulatory element of SEQ ID NO: 10 (wherein the GBA nucleotide sequence of SEQ ID

NO: 9 and the promoter element of SEQ ID NO: 10 are preferably operably linked). In

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such embodiments the GCase encoded by the GBA nucleotide sequence may be expressed

in human liver cells at least 1.1x, at least 1.2x, at least 1.3x, at least 1.4x, or at least 1.5x

higher. In an embodiment, a GCase polypeptide encoded by the GBA nucleotide sequence

is expressed in human liver cells at higher or non-statistically significant different levels

compared to a polypeptide encoded by an otherwise identical reference polynucleotide

comprising a GBA nucleotide sequence of SEQ ID NO: 9 operably linked to a promoter of

SEQ ID NO: 13, wherein the two polynucleotides are delivered to the cells in the same

way and in the same amounts.

In one embodiment, when the polynucleotide sequence comprising a GBA nucleotide

sequence is administered to a subject, or a non-human mammal such as a mouse, the

GCase is present in the serum of the subject or non-human animal at higher levels (for

example, at 4 or 8 or 12 weeks post-administration) compared to GCase encoded by an

otherwise identical nucleotide sequence comprising a GBA nucleotide sequence of SEQ ID

NO: 9 operably linked to a promoter element of SEQ ID NO: 12, 13 or 15, wherein the

polynucleotides comprising the GBA nucleotide are administered in the same way and in

the same amounts.

The skilled person may determine whether GCase is expressed from a given GBA

nucleotide sequence (for example, a codon-optimised GBA nucleotide sequence) at higher

levels compared to a reference sequence (for example, a wild type GBA nucleotide

sequence, such as SEQ ID NO: 9) by transducing some cells with a viral particle

comprising the GBA nucleotide sequence, and some cells with a particle comprising the

reference sequence. The cells may be cultured under conditions suitable for expressing the

GCase protein or fragment thereof encoded by the GBA nucleotide sequence, and the level

of expressed GCase protein can be compared. The level of expressed GCase protein can

be assessed using a fluorometric assay as described in the section entitled "GCase protein

or fragment thereof', or an thereof, or an ELISA ELISA using using aa GCase-specific GCase-specific antibody. antibody. Suitable Suitable cells cells include include

cultured human liver cells, such as Huh-7 cells.

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As discussed above, the presence of CpGs (i.e. CG dinucleotides) may reduce expression

efficiency. This is because CpGs may be methylated, and their methylation may lead to

gene silencing thereby reducing expression. Also, it is possible that high CpG content

could trigger a TLR response, increasing the risk of an anti-AAV immune response. For

this reason, it is preferred that the portion of the coding sequence that is codon-optimised

comprises a reduced number of CpGs compared to a corresponding portion of a reference

wild type GBA nucleotide sequence (such as SEQ ID NO: 9). In an embodiment, the

portion of the GBA nucleotide sequence that is codon-optimised (which may be all of the

GBA nucleotide sequence) comprises less than 40, less than 20, less than 10, or less than 5

CpGs. In an embodiment, the portion of the GBA nucleotide sequence that is codon-

optimised (which may be all of the GBA nucleotide sequence) comprises less than 5, less

than 4, less than 3, or less than 2 CpGs per 100 nts. In some embodiments, the portion of

the coding sequence that is codon-optimised is CpG-free, i.e. contains no (0) CG

dinucleotides.

In an embodiment, the portion of the GBA nucleotide sequence that is codon-optimised is

at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least

99.5%, at least 99.8%, or 100% identical to a fragment of at least 1000, at least 1200, at

least 1300, less than 1494, between 1000 and 1494, between 1300 and 1494, or around

1494 nucleotides of SEQ ID NO: 1-4. In an embodiment, the portion of the coding

sequence that is codon-optimised is at least 80%, at least 85%, at least 90%, at least 95%,

at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO:

1-4. In an embodiment, the portion of the GBA nucleotide sequence that is codon-

optimised is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least

99%, at least 99.5%, at least 99.8%, or 100% identical to a fragment of at least 1000, at

least 1200, at least 1300, less than 1494, between 1000 and 1494, between 1300 and 1494,

or around 1494 nucleotides of SEQ ID NO: 1. In an embodiment, the portion of the GBA

nucleotide sequence that is codon-optimised is at least 99.5%, at least 99.8%, or 100%

identical to a fragment of at least 1300 nucleotides of SEQ ID NO: 1. In an embodiment,

the portion of the coding sequence that is codon-optimised is at least 80%, at least 85%, at

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least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100%

identical identical to to SEQ SEQ ID ID NO: NO: 1. 1.

The present invention provides a polynucleotide comprising a GBA nucleotide sequence

that encodes a GCase protein or a fragment thereof and the GBA sequence comprises a

sequence sequence that that is is at at least least 95%, 95%, at at least least 98%, 98%, at at least least 99%, 99%, at at least least 99.5%, 99.5%, at at least least 99.8%, 99.8%, or or

100% identical to SEQ ID NO: 1. Optionally, the sequence that is at least 95%, at least

98%, at least 99%, at least 99.5%, or at least 99.8% identical to SEQ ID NO: 1 is codon-

optimised.

Portion of the coding sequence that is not codon-optimised

In an embodiment, the GBA nucleotide sequence comprises a portion that is not codon-

optimised. The portion that is not codon-optimised may be a contiguous portion.

As would be understood in the art, the portion that is not codon-optimised is therefore not

modified to include a greater number of favoured codons compared to the wild type

sequence. A contiguous non-codon-optimised polynucleotide sequence is a wild type

sequence.

Optionally, the portion that is not codon-optimised is at least 80, at least 90, at least 100, at

least 110, less than 200, less than 170, less than 140, or around 117 nucleotides. In some

embodiments, the portion that is not codon-optimised in a GBA nucleotide sequence is the

portion which encodes (corresponds to) all or a portion of the signal peptide. Optionally,

the portion that is not codon-optimised encodes all or a portion of a GCase signal peptide.

In some embodiments, the portion that is not codon-optimised in a GBA nucleotide

sequence is a portion having a sequence of SEQ ID NO: 17.

The polymucleotide polymicleotide may further comprise a transcription regulatory element

The polynucleotide may comprise a transcription regulatory element.

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In one embodiment, the transcription regulatory element is at least 80%, at least 85%, at

identical to SEQ ID NO: 10. In an embodiment, the polynucleotide comprises a

transcription regulatory element that is at least 98%, at least 99%, at least 99.5%, at least

99.8%, or 100% identical to SEQ ID NO: 10. Optionally, the polynucleotide comprises a

transcription regulatory element at least 98% identical to SEQ ID NO: 10. Optionally, the

polynucleotide comprises a transcription regulatory element of SEQ ID NO: 10.

Optionally, the polynucleotide comprises a transcription regulatory element consisting of

SEQ ID NO: 10.

In another embodiment, the transcription regulatory element is at least 80%, at least 85%,

at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or

100% identical to SEQ ID NO: 14. In an embodiment, the polynucleotide comprises a

99.8%, or 100% identical to SEQ ID NO: 14. Optionally, the polynucleotide comprises a

transcription regulatory element at least 98% identical to SEQ ID NO: 14. Optionally, the

polynucleotide comprises a transcription regulatory element of SEQ ID NO: 14.

SEQ ID NO: 14.

Any appropriate transcription regulatory element may be used, such as HLP2, HLP1, LP1,

HCR-hAAT, ApoE-hAAT, and LSP, which are all liver-specific transcription regulatory

elements. These transcription regulatory elements are described in more detail in the

following references: HLP1: McIntosh J. et al., Blood 2013 Apr 25, 121(17):3335-44;

LP1: Nathwani et al., Blood. 2006 April 1, 107(7): 2653-2661; HCR-hAAT: Miao et al.,

Mol Ther. 2000;1; 2000;1: 522-532; ApoE-hAAT: Okuyama et al., Human Gene Therapy, 7, 637-

645 (1996); and LSP: Wang et al., Proc Natl Acad Sci USA. US A.1999 1999March March30, 30,96(7): 96(7):

3906-3910.

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The transcription regulatory element may comprise a promoter and/or an enhancer, such as

the promoter element and/or enhancer element from HLP2, HLP1, LP1, HCR-hAAT,

ApoE-hAAT, and LSP. Each of these transcription regulatory elements comprises a

promoter, an enhancer, and optionally other nucleotides.

In an embodiment, the transcription regulatory element comprises an enhancer which is the

human apolipoprotein E (ApoE) hepatic locus control region (HCR; Miao et al (2000),

Molecular Therapy 1(6):522), or a fragment thereof. In an embodiment, the transcription

regulatory element comprises a fragment of the HCR enhancer which is a fragment of at

least 80, at least 90, at least 100, less than 192, between 80 and 192, between 90 and 192,

between 100 and 250, or between 117 and 192 nucleotides in length. Optionally, the

fragment of the HCR enhancer is between 100 and 250 nucleotides in length. In another

embodiment, the fragment of an HCR enhancer is a fragment of at least 150, at least 190,

at least 230, less than 400, between 150 and 400, between 190 and 370, between 230 and

340, 340, between between 250 250 and and 340, 340, or or around around 321 321 nucleotides nucleotides in in length. length. Optionally, Optionally, the the fragment fragment

of the HCR enhancer is between 250 and 340 nucleotides in length.

Suitable HCR enhancer element fragment are described in SEQ ID NOs: 11 and 16.

Optionally, the transcription regulatory element comprises an enhancer that is at least 80,

at least 90, at least 100, less than 192, between 80 and 192, between 90 and 192, between

100 and 250, or between 117 and 192 nucleotides in length and the enhancer comprises a

polynucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at

least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical SEQ ID NO: 11.

Optionally, the transcription regulatory element comprises an enhancer that is between 117

and 192 nucleotides in length and the enhancer comprises a polynucleotide sequence that is

at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical SEQ ID NO.

11. Optionally, the transcription regulatory element comprises an enhancer that is at least

80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at

least 99.8%, or 100% identical to a fragment of at least 90, at least 100, or at least 110

nucleotides of SEQ ID NO: 11. Optionally, the polynucleotide comprises an enhancer that

is at least 80%, at least 85%, at least 90%, at least 95% at least 98%, at least 99%, at least

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99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 11. Optionally, the

polynucleotide comprises an enhancer that is at least 98%, at least 99%, at least 99.5%, at

least 99.8%, or 100% identical to SEQ ID NO: 11. Optionally, the polynucleotide

comprises an enhancer of SEQ ID NO: 11. Optionally, the transcription regulatory element

comprises a fragment of an HCR enhancer that is equal to or less than 321 nucleotides,

equal to or less than 192 nucleotides or equal to or less than 117 nucleotides in length and

comprises SEQ ID NO: 11.

In another embodiment, the transcription regulatory element comprises an enhancer that is

at least 150, at least 190, at least 230, less than 400, between 150 and 400, between 190

and 370, between 230 and 340, between 250 and 340, or around 318 nucleotides in length

and the enhancer comprises a polynucleotide sequence that is at least 80%, at least 85%, at

identical SEQ ID NO: 16. Optionally, the transcription regulatory element comprises an

enhancer that is between 250 and 340 nucleotides in length and the enhancer comprises a

polynucleotide sequence that is at least 98%, at least 99%, at least 99.5%, at least 99.8%,

or 100% identical SEQ ID NO: 16. Optionally, the transcription regulatory element

comprises an enhancer that is at least 80%, at least 85%, at least 90%, at least 95%, at least

98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to a fragment of at

least 250 nucleotides of SEQ ID NO: 16. Optionally, the polynucleotide comprises an

enhancer that is at least 80%, at least 85%, at least 90%, at least 95% at least 98%, at least

99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 16. Optionally, the

least 99.8%, or 100% identical to SEQ ID NO: 16. Optionally, the polynucleotide

comprises an enhancer of SEQ ID NO: 16.

In an embodiment, the transcription regulatory element comprises a promoter which is a

human alpha-1 anti-trypsin promoter (A1AT; Miao et al (2000), Molecular Therapy

1(6):522), or a fragment thereof. Optionally, a fragment of an A1AT promoter which is at

least 100, at least 120, at least 150, at least 180, less than 255, between 100 and 255,

between 150 and 225, between 150 and 300, or between 180 and 255 nucleotides in length.

26

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Optionally, the fragment of an A1AT promoter is between 150 and 300 nucleotides in

length. In another embodiment, a fragment of an A1AT promoter which at least 200, at

least 250, at least 300, less than 500, between 200 and 500, between 250 and 500, or

between 350 and 450 nucleotides in length. Optionally, the fragment of an A1AT promoter

is between 350 and 450 nucleotides in length.

Suitable A1AT promoter fragments are described in SEQ ID NOs: 12 and 15. Optionally,

the transcription regulatory element comprises a promoter that is at least 100, at least 120,

at least 150, at least 180, less than 255, between 100 and 255, between 150 and 300, or

between 180 and 255 nucleotides in length and the promoter comprises a polynucleotide

sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least least

99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 12. Optionally, the

transcription regulatory element comprises a promoter that is between 180 and 255

nucleotides in length and the promoter comprises a polynucleotide sequence that is at least

98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 12.

Optionally, Optionally, the the polynucleotide polynucleotide comprises comprises aa promoter promoter that that is is at at least least 80%, 80%, at at least least 85%, 85%, at at

identical to a fragment of at least 100, at least 120, or at least 150 nucleotides of SEQ ID

NO: 12. Optionally, the polynucleotide comprises a promoter that is at least 80%, at least

85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%,

or 100% identical to SEQ ID NO: 12. Optionally, the polynucleotide comprises a

promoter that is at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100%

identical to SEQ ID NO: 12. Optionally, the polynucleotide comprises a promoter of SEQ

ID NO: 12. Optionally, the transcription regulatory element comprises a fragment of an

A1AT promoter that is equal to or less than 418 nucleotides, equal to or less than 255

nucleotides or equal to or less than 185 nucleotides in length and comprises SEQ ID NO:

12.

In another embodiment, the transcription regulatory element comprises a promoter that is

at least 200, at least 250, at least 300, less than 500, between 200 and 500, between 250

and 500, between 350 and 450, or around 418 nucleotides in length and the promoter

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comprises a polynucleotide sequence that is at least 80%, at least 85%, at least 90%, at

least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to

SEQ ID NO: 15. Optionally, the transcription regulatory element comprises a promoter

that is between 350 and 450 nucleotides in length and the promoter comprises a

or 100% identical to SEQ ID NO: 15. Optionally, the polynucleotide comprises a promoter

that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at

least 99.5%, at least 99.8%, or 100% identical to a fragment of at least 350 nucleotides of

SEQ ID NO: 15. Optionally, the polynucleotide comprises a promoter that is at least 80%,

at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least

99.8%, or 100% identical to SEQ ID NO: 15. Optionally, the polynucleotide comprises a

identical to SEQ ID NO: 15. Optionally, the polynucleotide comprises a promoter of SEQ

NO: 15. ID NO: 15.

If the polynucleotide is intended for expression in the liver, the promoter may be a liver-

specific promoter. Optionally, the promoter is a human liver-specific promoter.

A "liver-specific promoter" is a promoter that provides a higher level of expression in liver

cells compared to other cells in general. For example, the skilled person can determine

whether a promoter is a liver-specific promoter by comparing expression of the

polynucleotide in liver cells (such as Huh-7 cells) with expression of the polynucleotide in

cells from other tissues. If the level of expression is higher in the liver cells, compared to

the cells from other tissues, the promoter is a liver-specific promoter. Optionally, the

transcription regulatory element or the promoter is liver-specific if it promotes protein

expression at higher levels in liver cells compared to cells from at least one other organ or

tissue and the transcription regulatory element or the promoter promotes protein expression

in the cells from at least one other organ or tissue at a level less than 40%, less than 30%,

less than 25%, less than 15%, less than 10%, or less than 5% of the level that the

transcription regulatory element or the promoter promotes protein expression in liver cells.

Optionally, the cells from at least one other organ or tissue are at least one of kidney cells,

pancreatic pancreatic cells, cells, breast breast cells, cells, neuroblastoma neuroblastoma cells, cells, lung lung cells, cells, and and early early BB cells. cells. Optionally, Optionally,

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the cells from at least one other organ or tissue are kidney cells, pancreatic cells, breast

cells, neuroblastoma cells, lung cells, and early B cells. Optionally, the cells from at least

one other organ or tissue are at least one of HEK293T cells, PANC1 cells, BxPC-3 cells,

MCF7 cells, 1643 cells, MRC-9 cells, and 697 cells. Optionally, the cells from at least one

other organ or tissue are HEK293T cells, PANC1 cells, BxPC-3 cells, MCF7 cells, 1643

cells, MRC-9 cells, and 697 cells.

In one embodiment, the polynucleotide of the invention may provide for GCase to be

specifically expressed in the liver. In such examples, the polynucleotide may promote

substantially more GCase expression in liver cells than in at least one other tissue type or

organ. In one example, the polynucleotide of the invention which provides for GCase to

be specifically expressed in the liver comprises a liver-specific promoter.

Optionally, the polynucleotide of the invention may provide for GCase to be expressed at

higher levels in liver cells compared to cells from at least one other organ or tissue and

such that GCase is expressed in the one other organ or tissue at a level less than 40%, less

than 30%, less than 25%, less than 15%, less than 10%, or less than 5% of the level of

GCase expression in liver cells, when measured in the same assay.

pancreatic cells, breast cells, neuroblastoma cells, lung cells, and early B cells. Optionally,

cells, MRC-9 cells, and 697 cells.

A viral particle comprising the polynucleotide polymicleotide

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The invention further provides a viral particle comprising a recombinant genome

comprising polynucleotides of the invention. For the purposes of the present invention, the

term "viral particle" refers to all or part of a virion. For example, the viral particle

comprises a recombinant genome and may further comprise a capsid. The viral particle

may be a gene therapy vector. Herein, the terms "viral particle" and "vector" are used

interchangeably. For the purpose of the present application, a "gene therapy" vector is a

viral particle that can be used in gene therapy, i.e. a viral particle that comprises all the

required functional elements to express a transgene, such as a GBA nucleotide sequence, in

a host cell after administration.

Suitable viral particles include a parvovirus, a retrovirus, a lentivirus or a herpes simplex

virus. The parvovirus may be an adeno-associated virus (AAV). The viral particle is

preferably a recombinant adeno-associated viral (AAV) vector or a lentiviral vector. More

preferably, the viral particle is an AAV viral particle. The terms AAV and rAAV are used

interchangeably herein, unless context obviously suggests otherwise.

The genomic organization of all known AAV serotypes is very similar. The genome of

AAV is a linear, single-stranded DNA molecule that is less than about 5,000 nucleotides in

length. Inverted terminal repeats (ITRs) flank the unique coding nucleotide sequences for

the non-structural replication (Rep) proteins and the structural (VP) proteins. The VP

proteins (VP1, -2 and -3) form the capsid. The terminal ~145 nt (ITRs) are self-

SO that an energetically stable intramolecular duplex complementary and are organized so

forming a T-shaped hairpin may be formed. These hairpin structures function as an origin

for viral DNA replication, serving as primers for the cellular DNA polymerase complex.

Following wild type (wt) AAV infection in mammalian cells the Rep genes (i.e. encoding

Rep78 and Rep52 proteins) are expressed from the P5 promoter and the P19 promoter,

respectively, and both Rep proteins have a function in the replication of the viral genome.

A splicing event in the Rep ORF results in the expression of four Rep proteins (i.e. Rep 78, Rep78,

Rep68, Rep52 and Rep40). However, it has been shown that the unspliced mRNA,

encoding Rep78 and Rep52 proteins, in mammalian cells are sufficient for AAV vector

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production. Also in insect cells the Rep78 and Rep52 proteins suffice for AAV vector

production.

The recombinant viral genome of the invention may comprise ITRs. It is possible for an

AAV vector of the invention to function with only one ITR. Thus, the viral genome

comprises at least one ITR, but, more typically, two ITRs (generally with one either end of

the viral genome, i.e. one at the 5' end and one at the 3' end). There may be intervening

sequences between the polynucleotide of the invention and one or more of the ITRs. The

polynucleotide may be incorporated into a viral particle located between two regular ITRs

or located on either side of an ITR engineered with two D regions.

AAV sequences that may be used in the present invention for the production of AAV

vectors can be derived from the genome of any AAV serotype. Generally, the AAV

serotypes have genomic sequences of significant homology at the amino acid and the

nucleic acid levels, provide an identical set of genetic functions, produce virions which are

essentially physically and functionally equivalent, and replicate and assemble by

practically identical mechanisms. For the genomic sequence of the various AAV serotypes

and an overview of the genomic similarities see e.g. GenBank Accession number U89790;

GenBank Accession number J01901; GenBank Accession number AF043303; GenBank

Accession number AF085716; Chiorini et al, 1997; Srivastava et al, 1983; Chiorini et al,

1999; Rutledge et al, 1998; and Wu et al, 2000. AAV serotype 1, 2, 3, 3B, 4, 5, 6, 7, 8, 9,

10, 11 or 12 may be used in the present invention. The sequences from the AAV serotypes

may be mutated or engineered when being used in the production of gene therapy vectors.

Optionally, an AAV vector comprises ITR sequences which are derived from AAV1,

AAV2, AAV4 and/or AAV6. Preferably the ITR sequences are AAV2 ITR sequences.

Herein, the term AAVx/y refers to a viral particle that comprises genomic components

such as at least ITRs from AAVx (wherein X x is a AAV serotype number) and has the

capsid from AAVy (wherein y is the number of the same or different serotype). For

example, an AAV2/8 vector may comprise a portion of a viral genome, including the ITRs,

from an AAV2 strain, and a capsid from an AAV8 strain.

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In an embodiment, the viral particle is an AAV viral particle comprising a capsid. AAV

capsids are generally formed from three proteins, VP1, VP2 and VP3. The amino acid

sequence of VP1 comprises the sequence of VP2. The portion of VP1 which does not form

part of VP2 is referred to as VPlunique lunique oror VP1U. VP1U. The The amino amino acid acid sequence sequence ofof VP2 VP2

comprises the sequence of VP3. The portion of VP2 which does not form part of VP3 is

referred to as VP2unique or VP2U. Optionally, the viral particle comprises a liver-tropic

or CNS-tropic capsid. Whether a viral particle (capsid) is tropic for a particular tissue can

be evaluated for example by administering such a particle expressing a marker gene such

as luciferase and imaging in vivo at multiple time points (for example as described in

Zincarelli et al (2008), Molecular Therapy, 16:1073-1080). A particle driving strong

marker expression in liver or CNS tissues, respectively, especially if in contrast to lesser

expression in other tissues, would be considered liver- or CNS-tropic.

In some embodiments, a liver-tropic capsid can be an AAV3- or AAV3B-derived capsid.

Optionally, the liver-tropic capsid comprises a sequence at least 98%, at least 99%, or at

least 99.5% identical to a fragment of at least 600, at least 650, at least 700, between 600

and 736, between 650 and 736or between 700 and 736amino acids of SEQ ID NO: 19, 20,

or 24. Optionally, the liver-tropic capsid comprises a sequence at least 99% identical to

SEQ ID NO: 19. Optionally, the liver-tropic capsid comprises a sequence at least 99%

identical to SEQ ID NO: 20. Optionally, the liver-tropic capsid comprises a sequence at

least 99% identical to SEQ ID NO: 24. Optionally, the CNS tropic capsid comprises a

sequence at least 98%, at least 99%, at least 99.5% identical to a fragment of at least 600,

at least 650, at least 700, between 600 and 736, between 650 and 736or between 700 and

736amino acids of SEQ ID NO: 21. Optionally, the CNS-tropic capsid comprises a

sequence at least 99% identical to SEQ ID NO: 21. A viral particle of the invention may be be

a "hybrid" particle in which the viral ITRs and viral capsid are from different parvoviruses,

such as different AAV serotypes. Preferably, the viral ITRs and capsid are from different

serotypes of AAV, in which case such viral particles are known as transcapsidated or

pseudotyped. Likewise, the parvovirus may have a "chimeric" capsid (e. g., containing

WO wo 2020/161483 PCT/GB2020/050251 PCT/GB2020/050251

sequences from different parvoviruses, preferably different AAV serotypes) or a "targeted"

capsid (e.g., (e. g.,a adirected directedtropism). tropism).

In some embodiments, the recombinant AAV genome comprises intact ITRs, comprising

functional terminal resolution sites (TRS). Such an AAV genome may contain one or two

resolvable ITRs, i.e. ITRs containing a functional TRS at which site-specific nicking can

take place to create a free 3' hydroxyl group which can serve as a substrate for DNA

polymerase to unwind and copy the ITR. Preferably, the recombinant genome is single-

stranded (i.e., it is packaged into the viral particle in a single-stranded form). Optionally,

the recombinant genome is not packaged in self-complementary configuration, i.e. the

genome does not comprise a single covalently-linked polynucleotide strand with

substantial self-complementary portions that anneal in the viral particle. Alternatively, the the

recombinant genome may be packaged in "monomeric duplex" form. "Monomeric

duplexes" are described in WO 2011/122950. The genome may be packaged as two

substantially complementary but non-covalently linked polynucleotides which anneal in

the viral particle.

The viral particle may further comprise a poly A sequence sequence.The Thepoly polyA Asequence sequencemay maybe be

positioned downstream of the nucleotide sequence encoding a functional GCase protein.

The poly A sequence may be a bovine growth hormone poly A sequence (bGHpA - SEQ

ID NO: 23). The poly A sequence may be between 250 and 270 nucleotides in length.

The viral particle may further comprise an intron sequence, such as a viral intron sequence,

optionally an SV40 intron sequence (SEQ ID NO: 22).

In one embodiment, the viral particle comprises a polynucleotide sequence comprising a

promoter element, an intron sequence, such as an SV40 intron sequence, a GBA nucleotide

sequence, and a poly A sequence, such as the bGHpA sequence. In such embodiments,

the intron sequence, such as the SV40 intron sequence, may be located between the

promoter element and the GBA nucleotide sequence. In such embodiments, the poly A

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sequence, such as the bGHpA sequence, may be located downstream of the GBA

nucleotide sequence.

The viral particle of the invention optionally expresses GCase highly in host cells. For

example, on transduction in Huh-7 cells, the viral particle of the present invention

expresses GCase protein or a fragment thereof at a higher level compared to an otherwise

identical viral particle comprising a GBA nucleotide sequence of SEQ ID NO: 9

transduced at a comparable amount into a comparable population of Huh-7 cells.

Optionally, after transduction into a population of Huh-7 cells, the viral particle of the

present invention expresses GCase protein at a higher level than a viral particle comprising

a GBA nucleotide sequence of SEQ ID NO: 9 and a transcription regulatory element of

SEQ ID NO: 10 or a promoter of SEQ ID NO: 12. Optionally, after transduction into a

population of Huh-7 cells, the viral particle of the present invention expresses GCase

protein at a higher level than a comparable viral particle comprising a GBA nucleotide

sequence of SEQ ID NO: 9 and a transcription regulatory element of SEQ ID NO: 10 or a

promoter sequence of SEQ ID NO: 12 transduced at a comparable amount into a

comparable population of Huh-7 cells. Optionally, after transduction into a population of of

Huh-7 cells, the viral particle expresses GCase protein at comparable level (i.e. a non-

statistically significantly different level) to a viral particle comprising a GBA nucleotide

sequence of SEQ ID NO: 9 and a promoter element of SEQ ID NO: 13 transduced at a

comparable amount into a comparable population of Huh-7 cells. In such embodiments,

the term the term"comparable "comparableviral particle" viral refers particle to a viral refers particle to a viral that is that particle the same is as theansame AAV as an AAV

viral particle of the invention, except the comparable viral particle comprises a different

GBA nucleotide sequence and a different transcription regulatory element. Optionally, the the

comparable viral particle comprises the same transcription regulatory element as the AAV

viral particle of the invention. Optionally, the activity is assessed using a chromogenic

assay such as the fluorometric assay discussed above.

In one embodiment, provided is a viral particle comprising a polynucleotide sequence, the

polynucleotide sequence comprising:

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a) a GBA nucleotide sequence having at least 98% sequence identity to SEQ ID NO:

5, operably linked to:

b) a transcriptional regulatory sequence having at least 98% sequence identity to SEQ

ID NO: 14;

wherein the viral particle further comprises a capsid having at least 98% identity to SEQ

ID NO: 20.

polynucleotide sequence comprising:

5, operably linked to:

ID NO: 10;

ID NO: 20.

Compositions, methods and uses

In a further aspect of the invention, there is provided a composition comprising the

polynucleotide or vector/viral particle of the invention and a pharmaceutically acceptable

excipient.

The pharmaceutically acceptable excipients may comprise carriers, diluents and/or other

medicinal agents, pharmaceutical agents or adjuvants, etc. Optionally, the

pharmaceutically acceptable excipients comprise saline solution. Optionally, the

pharmaceutically acceptable excipients comprise human serum albumin.

This invention further provides a method of expressing the GBA nucleotide sequence and

achieving a stable GCase activity in a subject and/or providing greater GCase

bioavailability in a subject compared to the bioavailability from GCase enzyme

replacement therapy, wherein the bioavailability is measured over a period of 2 weeks

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from administration, wherein the method comprises administration of a polynucleotide,

viral particle or composition of the invention to a subject.

The invention further provides a polynucleotide, vector/viral particle or composition of the

invention for use in a method of treatment. Optionally the method of treatment comprises

administering an effective amount of the polynucleotide or vector/viral particle of the

invention to a patient.

The invention further provides a method of treatment comprising administering an

effective amount of the polynucleotide or vector/viral particle of the invention to a patient.

The invention further provides use of the polynucleotide, vector/viral particle or

composition of the invention in the manufacture of a medicament for use in a method of

treatment. Optionally the method of treatment comprises administering an effective

amount of the polynucleotide or vector/viral particle of the invention to a patient.

Optionally the method of treatment is a gene therapy. A "gene therapy" involves

administering a vector/viral particle of the invention that is capable of expressing a

transgene (such as a GBA nucleotide sequence) in the host to which it is administered.

Optionally, the method of treatment is a method of treating diseases associated with a

GCase deficiency. As discussed above, GCase deficiency may lead to accumulation of

glucocerebrosides in macrophages that infiltrate many vital organs which can cause a

variety of diseases including synucleopathies (as discussed in WO08/144591) or

Parkinson's disease. Parkinson's Optionally, disease. the method Optionally, of treatment the method is a method of treatment is aofmethod treating of treating

Parkinson's disease or a synucleopathy.

Optionally, the method of treatment is a method of treating a lysosomal storage disorder

such as Gaucher disease (GD), for example GD type I, type II or type III. Preferably, the

lysosomal storage disorder is characterised by bruising, fatigue, anemia, low blood platelet

count and enlargement of the liver and spleen. Optionally, the method of treatment is a

method of treating GD, for example GD type I. In some embodiments, the patient is a

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patient suffering from GD, for example GD type I. Optionally the patient has antibodies or

inhibitors to recombinant GCase (for example imiglucerase, velaglucerase alfa or

taliglucerase alfa) with which the patient has previously been treated as part of an enzyme

replacement therapy. Optionally, the polynucleotide and/or vector/viral particle is

administered intravenously. Optionally, the polynucleotide and/or vector/viral particle is

for administration only once (i.e. a single dose) to a patient.

When GD is "treated" in the above method, this means that one or more symptoms of GD

type I are ameliorated. It does not mean that the symptoms of GD type I are completely

remedied SO so that they are no longer present in the patient, although in some methods, this

may be the case. Thus, in all instances the term "treatment" can be replaced with the term

"amelioration". The method of treatment may result in one or more of the symptoms of

GD type I being less severe than before treatment. Optionally, relative to the situation pre-

administration, the method of treatment results in an increase in the amount/concentration

of circulating GCase in the blood of the patient, and/or the overall level of GCase activity

detectable within a given volume of blood and/or the macrophages of the patient. In one

embodiment, relative to the situation pre-administration, the method of treatment results in

one or more of: an increase in haemoglobin concentration; an increase in platelet count; a

decrease in spleen size; a decrease in liver size.

In addition, the methods of the invention may "prevent" diseases such as Gaucher disease.

Gaucher disease is generally associated with an accumulation of glucocerebrosidases in

various tissues, and if the methods of the invention are carried out on young subjects (such

as teenagers, young adults, children or babies) it should be possible to prevent Gaucher

disease from establishing. Accordingly, in all instances the term "treatment" may be

replaced with the term "prevention".

A "therapeutically effective amount" refers to an amount effective, at dosages and for

periods of time necessary, to achieve the desired therapeutic result, such as raising the level

of functional GCase in a subject (so as to lead to functional GCase production at a level

sufficient to ameliorate the symptoms of GD, for example GD type I).

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Optionally, the vector/viral particle is administered at a dose of less than 1 X 1011, 10¹¹, less than

1 x 10 ¹2, less 10¹², less than than 55 XX 10¹², 10 12, less less than than 2 2 X X 1012, 10¹², less less than than 1.5 1.5 X X 1012, 10¹², less less than than 3 3 X X 1012, 10¹², less less

than 10 1 x1 10¹³, 3, less than less X 10 than 2 x13, or less 10¹³, than or less 3 x 310 than x 13 vector 10¹³ genomes vector per genomes kg kg per of of weight of of weight

patient. Optionally, the dose of vector/viral particle that is administered is selected such

that the subject expresses GCase at an level of 10%-90%, 20%-80%, 30%-70%, 25%-50%,

20%-150%, 30%-140%, 40%-130%, 50%-120%, 60%-110% or 70%-100% of the level of

a healthy subject who does not suffer from GD.

Optionally, a patient administered the polynucleotide, viral particle or composition may

have a GCase activity level of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6,

at least 7, at least 8, or at least 9 umol/h/ml. µmol/h/ml. Optionally, the GCase activity is measured

using a fluorometric substrate which is specific for GCase. Optionally, the GCase activity

is measured fluorometrically with 4-Methylumbelliferyl-B-D-glucopyranoside 4-Methylumbelliferyl-ß-D-glucopyranoside (4MU-Glc)

as a substrate. Optionally, the GCase activity is measured in the serum, plasma,

macrophages, spleen, liver and/or bone marrow of the subject.

In one embodiment, GCase activity may be determined fluorometrically with 4-

Methylumbelliferyl-B-D-glucopyranoside (4MU-Glc) Methylumbelliferyl-ß-D-glucopyranoside (4MU-Glc) as as aa substrate substrate as as follows: follows: (1) (1) serum serum

samples are collected or tissues (liver, spleen, bone marrow) are harvested and snap frozen

and lysed; (2) the tissue lysate or serum/plasma sample are mixed in 50 mM Sodium

Citrate, 25 mM Taurocholate, pH=5.75, 6 mM 4MU-Glc, for 30 min at 37°; 37°C;(3) (3)the the

reaction is stopped by adding one volume (100 ul) µ1) of stop solution (0.5 M Glycine, 0.3 M

NaOH, pH 10.0); (4) relative fluorescence levels (RFU) are evaluated with a Spectramax

I3X (Molecular devices) using excitation and emission wavelengths of 365 nm and 445

nm, respectively and fluorescence levels were then converted to nanomoles/h/mL based on

a 4-Methylumbelliferone (4-MU, Sigma-Aldrich) standard curve.

have a greater GCase activity level at least 5, at least 10, at least 15, at least 20, at least 25,

at least 30, or at least 35 weeks after administration when compared to the activity

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measured in a subject administered an effective dose of a GCase enzyme replacement

therapy, when measured in the same assay at the same time point after administration.

have a GCase activity level greater by 10 fold, 20 fold, 50 fold, 100 fold or 1000 fold at

least 5, at least 10, at least 15, at least 20, at least 25, at least 30, or at least 35 weeks after

administration when compared to the activity measured in a subject administered an

effective dose of a GCase enzyme replacement therapy, when measured in the same assay

at the same time point after administration.

Optionally, the dose of vector/viral particle that is administered is selected such that there

is a greater GCase bioavailability to the subject when compared to the bioavailability from

GCase enzyme replacement therapy. Bioavailability may be measured (e.g. estimated or

calculated) through any known method in the art. GCase bioavailability may be measured

in the serum, macrophages, spleen, liver and/or bone marrow of the subject. In one

example, bioavailability may be estimated using the area under the curve ("AUC") method

according to Example 8. In one example, bioavailability may be estimated by estimating

the total GCase activity available in the serum, plasma, macrophages, spleen, liver and/or

bone marrow of the subject. Optionally, it is calculated over a defined time period, and

refers to the total activity or concentration of GCase during that time period. Optionally,

the GCase activity is measured using a fluorometric substrate which is specific for GCase.

Optionally, the GCase activity is measured fluorometrically with 4-Methylumbelliferyl-B-

D-glucopyranoside D-glucopyranoside (4MU-Glc) (4MU-Glc) as as aa substrate. substrate. Optionally, Optionally, the the GCase GCase activity activity is is measured measured

in the serum, plasma, macrophages, spleen, liver and/or bone marrow of the subject.

Optionally the GCase activity is measured in the white blood cells of the subject.

Optionally, the bioavailability is measured over a period of 2 weeks from administration.

Optionally, the bioavailability is measured over a period of 5 weeks from administration.

Optionally, the bioavailability is measured in serum. In one example, a greater GCase

bioavailability in the subject is achieved over a period of at least 5, at least 10, at least 15,

at least 20, at least 25, at least 30, or at least 35 weeks after administration when compared

to the bioavailability measured in a subject administered an effective dose of a GCase

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enzyme replacement therapy, when measured in the same assay at the same time point

after administration.

Optionally, a patient (for example, a patient suffering from a disease or condition

associated with GCase deficiency) administered the polynucleotide, viral particle or

composition of the invention may have reduced hexosylceramide and/or

hexosylsphingosine levels after administration, preferably when the hexosylceramide

and/or hexosylsphingosine levels are measured 6 weeks, 8 weeks, 10 weeks or 12 weeks

after administration. The hexosylceramide and/or hexosylsphingosine levels may be

reduced by 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or

more, 2 to 3 times, 2 to 4 times, 2 to 5 times, 2 to 6 times, or 3 to 5 times when compared

to the the (starting) (starting) hexosylceramide hexosylceramide and/or and/or hexosylsphingosine hexosylsphingosine levels atlevels at of the time the time of

administration of the polynucleotide, viral particle or composition of the invention. For

example, after administration of the polynucleotide viral particle or composition of the

invention (for example 6 weeks, 8 weeks, 10 weeks or 12 weeks after administration), the

hexosylceramide and/or hexosylsphingosine levels in the patient may be 50% or less, 40%

or less, 30% or less, 25% or less, 20% or less when compared to the (starting)

hexosylceramide and/or hexosylsphingosine levels at the time of administration of the

polynucleotide, viral particle or composition of the invention. Optionally, the patient may

have increased hexosylceramide and/or hexosylsphingosine levels when compared to a

healthy subject or a subject who does not have a disease or condition associated with

GCase deficiency. For example, the hexosylceramide and/or hexosylsphingosine levels are

measured in the spleen, liver and/or bone marrow of the patient/subject. The

hexosylceramide and/or hexosylsphingosine levels may be measured in the serum and/or

white blood cells (e.g. macrophages) of the patient/subject. Methods of measuring

hexosylceramide and/or hexosylsphingosine levels are known in the art, and the levels of

hexosylceramide and/or hexosylsphingosine are preferably measured using mass

spectrometry (LC/MS analysis), for example by the method described in example 9.

Optionally, the reduction of hexosylceramide and/or hexosylsphingosine levels (for

example in the serum, white blood cells (e.g. macrophages), spleen, liver and/or bone

marrow of the patient/subject) are greater than the reduction achieved from GCase enzyme

WO wo 2020/161483 PCT/GB2020/050251 PCT/GB2020/050251

replacement therapy, preferably when the hexosylceramide and/or hexosylsphingosine

levels are measured after at least 6 weeks, at least 8 weeks, at least 10 weeks or at least 12

weeks after the start of treatment. For example levels after at least 6 weeks (e.g. at 6

weeks), at least 8 weeks (e.g. at 8 weeks), at least 10 weeks (e.g. at 10 weeks) or at least 12

weeks (e.g. at 12 weeks) from administration of the polynucleotide, viral particle or

composition of the invention may be compared to levels after at least 6 weeks (e.g. at 6

weeks (e.g. at 12 weeks), respectively, from the first administration of GCase enzyme

replacement therapy. As a particular example, hexosylceramide and/or

hexosylsphingosine levels may be measured after at least 12 weeks (e.g. at 12 weeks) after

administration of the polynucleotide, viral particle or composition of the invention and

compared to the levels measured at least 12 weeks (e.g. at 12 weeks) after first

administration of GCase enzyme replacement therapy. Preferably, the levels of

hexosylceramide and/or hexosylsphingosine are measured in the same assay at the same

time point after administration. Optionally, the GCase enzyme replacement therapy may

be administered every two weeks. Optionally, the reduction of hexosylceramide levels in

the subject (or patient) after administration of the polynucleotide, viral particle or

composition of the invention are such that the hexosylceramide levels (for example in the

serum, white blood cells (e.g. macrophages), liver and/or spleen) are no more than 200%, 200%

150%, or 125% of the hexosylceramide levels measured in a healthy subject or a subject

not suffering from a disease or condition associated with GCase deficiency. In one

example, a reduction in hexosylceramide and/or hexosylsphingosine levels may represent a

reduction in glucosylceramide and/or glucosylsphingosine levels, respectively. For

example, a reduction in hexosylceramide may represent a reduction in glucosylceramide.

As a further example, a reduction in hexosylsphingosine levels may represent a reduction

in glucosylsphingosine levels.

In one example, a reduction in hexosylceramide and/or hexosylsphingosine levels is a

reduction in glucosylceramide and/or glucosylsphingosine respectively. In other words, a

patient (for example, a patient suffering from a disease or condition associated with GCase

deficiency) administered the polynucleotide, viral particle or composition of the invention

WO wo 2020/161483 PCT/GB2020/050251 PCT/GB2020/050251

may have reduced glucosylceramide and/or glucosylsphingosine levels after

administration, preferably when the glucosylceramide and/or glucosylsphingosine levels

are measured 6 weeks, 8 weeks, 10 weeks or 12 weeks after administration. The

glucosylceramide and/or glucosylsphingosine levels may be reduced by 2 times or more, 3

times or more, 4 times or more, 5 times or more, 6 times or more, 2 to 3 times, 2 to 4 times,

2 to 5 times, 2 to 6 times, or 3 to 5 times when compared to the (starting) glucosylceramide

and/or glucosylsphingosine levels at the time of administration of the polynucleotide, viral

particle or composition of the invention. For example, after administration of the

polynucleotide viral particle or composition of the invention (for example 6 weeks, 8

weeks, 10 weeks or 12 weeks after administration), the glucosylceramide and/or

glucosylsphingosine levels glucosylsphingosine in the levels in patient may bemay the patient 50% be or 50% less,or 40% or less, less, 40% 30% or less, or less, 30% or less,

25% or less, 20% or less when compared to the (starting) glucosylceramide and/or

glucosylsphingosine levels at the time of administration of the polynucleotide, viral

particle or composition of the invention. Optionally, the patient may have increased

glucosylceramide and/or glucosylsphingosine levels when compared to a healthy subject or

a subject who does not have a disease or condition associated with GCase deficiency. For

example, the glucosylceramide and/or glucosylsphingosine levels are measured in the

spleen, liver and/or bone marrow of the patient/subject. The glucosylceramide and/or

glucosylsphingosine levels may be measured in the serum and/or white blood cells (e.g.

macrophages) of the patient/subject. Methods of measuring glucosylceramide and/or

glucosylsphingosine levels are known in the art, and the levels of glucosylceramide and/or

glucosylsphingosine are preferably measured using mass spectrometry (LC/MS analysis),

for example by the method described in example 9. Optionally, the reduction of

glucosylceramide and/or glucosylsphingosine levels (for example in the serum, white

blood blood cells cells(e.g. macrophages), (e.g. spleen, macrophages), liver liver spleen, and/or and/or bone marrow boneofmarrow the patient/subject) of the patient/subject)

are greater than the reduction achieved from GCase enzyme replacement therapy,

preferably when the glucosylceramide and/or glucosylsphingosine levels are measured

after at least 6 weeks, at least 8 weeks, at least 10 weeks or at least 12 weeks after the start

of treatment. For example levels after at least 6 weeks (e.g. at 6 weeks), at least 8 weeks

(e.g. at 8 weeks), at least 10 weeks (e.g. at 10 weeks) or at least 12 weeks (e.g. at 12

weeks) from administration of the polynucleotide, viral particle or composition of the

WO wo 2020/161483 PCT/GB2020/050251 PCT/GB2020/050251

invention may be compared to levels after at least 6 weeks (e.g. at 6 weeks), at least 8

weeks (e.g. at 8 weeks), at least 10 weeks (e.g. at 10 weeks) or at least 12 weeks (e.g. at 12

weeks), respectively, from the first administration of GCase enzyme replacement therapy.

As a particular example, glucosylceramide and/or glucosylsphingosine levels may be

measured after at least 12 weeks (e.g. at 12 weeks) after administration of the

polynucleotide, viral particle or composition of the invention and compared to the levels

measured at least 12 weeks (e.g. at 12 weeks) after first administration of GCase enzyme

replacement therapy. Preferably, the levels of glucosylceramide and/or

glucosylsphingosine are measured in the same assay at the same time point after

administration. Optionally, the GCase enzyme replacement therapy may be administered

every two weeks. Optionally, the reduction of glucosylceramide levels in the subject (or

patient) after administration of the polynucleotide, viral particle or composition of the

invention are such that the glucosylceramide levels (for example in the serum, white blood

cells (e.g. macrophages), liver and/or spleen are no no ) are more than more 200%, than 150%, 200%, or or 150%, 125% of of 125%

the glucosylceramide levels measured in a healthy subject or a subject not suffering from a

disease or condition associated with GCase deficiency.

Optionally a patient (for example, a patient suffering from a disease or condition associated

with GCase deficiency) administered the polynucleotide, viral particle or composition of

the invention may show a reduced number of storage cells and/or activated macrophages in

the liver after administration, preferably when the cells are counted after at least 6 weeks

(e.g. at 6 weeks), at least 8 weeks (e.g. at 8 weeks), at least 10 weeks (e.g. at 10 weeks) or

at least 12 weeks (e.g. at 12 weeks) after administration. Reduction in the number of

storage cells and/or activated macrophages in the liver may be an indication of reduced

inflammation and thus therapeutic benefit. The number of activated macrophages may be

indicated or estimated by measuring the number of CD68positive cells. Identifying storage indicated or estimated by measuring the number of cells. Identifying storage cells and CD68 positive cells can be performed by methods known in the art, for example the

methods described in example 9.

A "GCase enzyme replacement therapy" may refer to any therapy which comprises the

administration of a GCase polypeptide to a subject. The GCase polypeptide may be wild

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type, such as a GCase polypeptide having the amino acid sequence of SEQ ID NO: 25.

The GCase polypeptide may be administered at any suitable dose, optionally at a dose of

between 40 and 100, between 50 and 80, between 60 and 70, or around 60 U/kg BW. The

GCase polypeptide may be administered through any appropriate route, optionally

administered through intravenous injection or subcutaneous injection.

A GCase activity level of at least X% (e.g. at least 20%) refers to a GCase activity level

that is at least X% (e.g. 20%) of the normal GCase level range as measured from a sample

of e.g. the spleen or bone marrow. The person skilled in the art would readily understand

what is meant by reference to a %-of-normal GCase activity level, which is determined in

routine clinical practice by e.g. comparison to a control sample from a healthy subject.

The term "stable GCase activity" or "stable GCase activity level" refers to a GCase

activity level that maintains at or above a certain level for a continuous period of at least 5

weeks. In other words, the activity may fluctuate above said activity level but is still said

to be stable as long as it remains above the stated minimum threshold. In some

embodiments, the GCase activity level maintains at or above a certain level for a

continuous period of at least 10, at least 15, at least 20, at least 30, at least 40, or at least 50

weeks. For example, a patient has a stable GCase activity level of at least 20% if the

activity level maintains at at least 20% for a continuous period of at least 5 weeks. In such

an example, the GCase activity level may continue to be at at least 20% following the at

least 5 weeks and thus maintains at at least 20% for a cumulative continuous period of at

least 10, at least 15, at least 20, at least 30 or at least 40, or at least 50 weeks. A patient has

a stable GCase activity level if the GCase activity level maintains at or above a certain

level for a continuous period of at least 5 weeks. Optionally, a patient administered with

the polynucleotide, viral particle or composition may have a stable GCase activity level of

at least 20%, at least 25%, at least 30%, at least 35%, at least 40% or at least 50% relative

to the GCase activity of a healthy subject. Optionally, a patient administered with the

polynucleotide, viral particle or composition may have a stable GCase activity level of at

least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8 or at least 9

umol/h/ml. µmol/h/ml. Optionally, the GCase activity is measured using a fluorometric substrate

WO wo 2020/161483 PCT/GB2020/050251 PCT/GB2020/050251

which is specific for GCase. Optionally, the GCase activity is measured fluorometrically

with 4-Methylumbelliferyl-B-D-glucopyranoside (4MU-Glc) as 4-Methylumbelliferyl--D-glucopyranoside (4MU-Glc) as aa substrate. substrate. Optionally, Optionally, the the

GCase activity is measured in the serum, macrophages, spleen, liver and/or bone marrow

of the subject.

Optionally, the GCase activity level is stable after at least 5 weeks, at least 10 weeks, at

least 15 weeks, at least 20 weeks, at least 30 weeks, at least 40 weeks, or at least 50 weeks

from administration of the polynucleotide, viral particle or composition. For example,

where a patient has a stable GCase activity level of at least 20% after at least 5 weeks from

when the patient is administered with the polynucleotide, viral particle or composition,

there is a GCase activity level of at least 20% that maintains at at least 20% for a

continuous period of at least 5, at least 10, at least 15, at least 20, at least 30 or at least 40,

or at least 50 weeks following the initial at least 5 weeks from administration.

Optionally, the GCase activity level is at or above a certain level (e.g. 20%, 25%, 30%,

35%, or 40%; and/or at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least

7, at least 8 or at least 9 umol/h/ml) µmol/h/ml) at a time point at least 5, at least 10, at least 20, at least

30, at least 40 or at least 50 weeks after administration of the polynucleotide, viral particle

or composition. For example, the GCase activity level is at or above a certain level (e.g.

20%, 25%, 30%, 35%, or 40%; and/or at least 1, at least 2, at least 3, at least 4, at least 5, at

least 6, at least 7, at least 8 or at least 9 umol/h/ml) µmol/h/ml) at a time point of around 4, 5, 6, 7, 8, 9,

10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,

34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 or 52 weeks after

administration of the polynucleotide, viral particle or composition.

The invention will now be described with reference to the following examples, which are

merely illustrative and should not in any way be construed as limiting the scope of the

present invention.

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Sequence Listing

Sequence identity number Sequence description 1 Codon-optimised GBA Codon-optimised nucleotide GBA sequence nucleotide from from sequence FLF- FLF- PL28, without signal peptide portion 2 Codon-optimised GBA nucleotide sequence from FLF- PL21, without signal peptide portion 3 Codon-optimised GBA nucleotide sequence from FLF- PL30, without signal peptide portion 4 Codon-optimised GBA nucleotide sequence from FLF- PL36, without signal peptide portion 5 Codon-optimised GBA nucleotide sequence from FLF- PL28, with signal peptide portion 6 Codon-optimised GBA nucleotide sequence from FLF- PL21, with signal peptide portion 7 Codon-optimised GBA nucleotide sequence from FLF- PL30, with signal peptide portion 8 Codon-optimised GBA Codon-optimised nucleotide GBA sequence nucleotide from from sequence FLF- FLF- PL36, with signal peptide portion

9 Wild type human GBA nucleotide sequence with signal peptide (from GenBank NM 000157.3) (000157.3) 10 LSP-S transcription regulatory element 11 HCR enhancer portion of LSP-S 12 A1AT promoter portion of LSP-S 13 CAG promoter 14 LSP-L transcription regulatory element 15 A1AT promoter portion of LSP-L 16 HCR enhancer portion of LSP-L 17 17 Wild type GBA nucleotide sequence corresponding to signal peptide

18 Wild type GCase polypeptide sequence of signal peptide 19 Polypeptide sequence of liver-tropic capsid

20 Polypeptide sequence of liver-tropic capsid 21 21 Polypeptide sequence of CNS-tropic capsid

22 Nucleotide sequence of SV40 intron

23 Nucleotide sequence of bovine growth hormone poly A

sequence 24 Polypeptide sequence of liver-tropic capsid

25 Polypeptide sequence of wild type human GCase

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Aspects Aspects of of the the invention invention

The invention is further described in the following aspects.

1. 1. A polynucleotide comprising a GBA nucleotide sequence, wherein the GBA

nucleotide sequence encodes a B-Glucocerebrosidase (GCase) protein -Glucocerebrosidase (GCase) protein or or fragment fragment

thereof and wherein at least a portion of the GBA nucleotide sequence is not wild

type.

2. The polynucleotide of aspect 1, wherein the portion of the GBA nucleotide sequence

that is not wild type is codon-optimised.

3. The polynucleotide of aspect 1 or 2, wherein the GBA nucleotide sequence encodes a

GCase protein or a fragment thereof and comprises a sequence that is at least 95%, at

least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or

100% identical to a nucleotide sequence of any one of SEQ ID NO: 1-8.

4. 4. The polynucleotide of any one of aspects 1 to 3, wherein the GBA nucleotide

sequence comprises a sequence of SEQ ID NO: 1 or a variant of SEQ ID NO: 1

encoding a GCase protein having GCase activity.

5. The polynucleotide of aspect 4, wherein the variant of SEQ ID NO: 1 is identical to

SEQ ID NO: 1 except that it comprises nucleotide substitutions such that the GCase

protein has 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, or up

to 10 amino acid substitutions relative to the wild type GCase amino acid sequence

of SEQ ID NO: 25.

6. The polynucleotide of aspect 4 or 5, wherein the variant of SEQ ID NO: 1 has 1, up

to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 20, or

up to 30 nucleotide substitutions relative to the sequence of SEQ ID NO: 1.

WO wo 2020/161483 PCT/GB2020/050251

7. The polynucleotide of any one of aspects 4 to 6, wherein the variant of SEQ ID NO:

1 has 1, up to 2, up to 3, up to 4, up to 5, or up to 6 nucleotide substitutions relative

to the sequence of SEQ ID NO: 1.

8. The polynucleotide of any one of aspects 4 to 7, wherein the variant of SEQ ID NO:

1 has has up uptoto4 4nucleotide nucleotide substitutions substitutions relative relative to the to the sequence sequence of SEQ IDof NO:SEQ 1 ID NO: 1

and/or encodes a GCase protein having up to 3 amino acid substitutions relative to

the wild type amino acid GCase sequence of SEQ ID NO: 25.

9. The polynucleotide of any one of aspects 4 to 8, wherein the variant of SEQ ID NO:

1 has up to 3 nucleotide substitutions relative to the sequence of SEQ ID NO: 1

and/or encodes a GCase protein having up to 2 amino acid substitutions relative to

the wild type GCase amino acid sequence of SEQ ID NO: 25.

10. The polynucleotide of any one of aspects 4 to 9, wherein the variant of SEQ ID NO:

1 has 1 nucleotide substitution relative to the sequence of SEQ ID NO: 1 and/or

encodes a GCase protein having up to 1 amino acid substitution relative to the wild

type GCase amino acid sequence of SEQ ID NO: 25.

11. 11. The Thepolynucleotide polynucleotideofofany anyone oneofofaspects aspects1 1toto3,3,wherein whereinthe theGBA GBAnucleotide nucleotide

sequence comprises a sequence of SEQ ID NO: 5 or a variant of SEQ ID NO: 5

encoding a GCase protein having GCase activity.

12. 12. The Thepolynucleotide polynucleotideofofaspect aspect11, 11,wherein whereinthe thevariant variantofofSEQ SEQIDIDNO: NO:5 5isisidentical identicaltoto

SEQ ID NO: 5 except that it comprises nucleotide substitutions such that the GCase

to 10 amino acid substitutions relative to the wild type GCase sequence of SEQ ID

NO: 25.

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13. The polynucleotide of aspect 11 or 12, wherein the variant of SEQ ID NO: 5 has 1,

up up to to 2, 2, up up to to 3, 3, up up to to 4, 4, up up to to 5, 5, up up to to 6, 6, up up to to 7, 7, up up to to 8, 8, up up to to 9, 9, up up to to 10, 10, up up to to 20, 20,

or up to 30 nucleotide substitutions relative to the sequence of SEQ ID NO: 5.

14. The polynucleotide of any one of aspects 11 to 13, wherein the variant of SEQ ID

NO: 5 has 1, up to 2, up to 3, up to 4, up to 5, or up to 6 nucleotide substitutions

relative to the sequence of SEQ ID NO: 5.

15. 15. The Thepolynucleotide polynucleotideofofany anyone oneofofaspects aspects1111toto14, 14,wherein whereinthe thevariant variantofofSEQ SEQIDID

NO: 5 has up to 4 nucleotide substitutions relative to the sequence of SEQ ID NO: 5

the wild type GCase amino acid sequence of SEQ ID NO: 25.

16. The polynucleotide of any one of aspects 11 to 15, wherein the variant of SEQ ID

NO: 5 has up to 3 nucleotide substitutions relative to the sequence of SEQ ID NO: 5

the wild type GCase amino acid sequence of SEQ ID NO: 25.

17. 17. The Thepolynucleotide polynucleotideofofany anyone oneofofaspects aspects1111toto16, 16,wherein whereinthe thevariant varianthas has1 1

nucleotide substitution relative to the sequence of SEQ ID NO: 5 and/or encodes a

GCase protein having up to 1 amino acid substitution relative to the wild type GCase

amino acid sequence of SEQ ID NO: 25.

18. 18. The Thepolynucleotide polynucleotideofofany anyone oneofofthe thepreceding precedingaspects, aspects,wherein whereinthe theGBA GBAnucleotide nucleotide

sequence encodes a GCase protein having 1, up to 2, up to 3, up to 4, or up to 5

amino acid substitutions relative to the wild type GCase amino acid sequence of SEQ

ID NO: 25.

19. The polynucleotide of any one of the preceding aspects, wherein the GBA nucleotide

sequence encodes a GCase protein having up to 3 amino acid substitutions relative to

the wild type GCase amino acid sequence of SEQ ID NO: 25.

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20. The polynucleotide of any one of the preceding aspects, wherein the GBA nucleotide

sequence encodes a GCase protein having up to 2 amino acid substitutions relative to

the wild type GCase amino acid sequence of SEQ ID NO: 25.

21. 21. The polynucleotide The ofof polynucleotide any one any ofof one the preceding the aspects, preceding wherein aspects, the wherein GBA the nucleotide GBA nucleotide

sequence encodes a variant GCase protein having up to 1 amino acid substitution

relative to the wild type GCase amino acid sequence of SEQ ID NO: 25.

22. A polynucleotide comprising a GBA nucleotide sequence, wherein the GBA

nucleotide sequence encodes a GCase protein or a fragment thereof and comprises a

sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at

least 99.5%, at least 99.8%, or 100% identical to a fragment of at least 1000, at least

1200, at least 1300, less than 1494, less than 1611, between 1000 and 1494, between

1000 and 1611, between 1300 and 1494, between 1300 and 1611, around 1494, or

around 1611 nucleotides of any one of SEQ ID NO: 1-8.

23. Thepolynucleotide 23. The polynucleotideofofany anyone oneofofthe thepreceding precedingaspects, aspects,wherein whereinthe theGBA GBAnucleotide nucleotide

sequence comprises a sequence that is at least 98% identical to a fragment of at least

1300 nucleotides of any one of SEQ ID NO: 1-8.

24. Thepolynucleotide 24. The polynucleotideofofany anyone oneofofthe thepreceding precedingaspects, aspects,wherein whereinthe theGBA GBAnucleotide nucleotide

sequence comprises a sequence that is at least 99% identical to a fragment of at least

1300 nucleotides of any one of SEQ ID NO: 1-8.

25. Thepolynucleotide 25. The polynucleotideofofany anyone oneofofthe thepreceding precedingaspects, aspects,wherein whereinthe theGBA GBAnucleotide nucleotide

sequence comprises a sequence that is at least 98% identical to a nucleotide sequence

of any one of SEQ ID NO: 1-8.

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26. The polynucleotide of any one of the preceding aspects, wherein the GBA nucleotide

sequence comprises a sequence that is at least 99% identical to a nucleotide sequence

of any one of SEQ ID NO: 1-8.

27. 27. The polynucleotide The ofof polynucleotide any one any ofof one the preceding the aspects, preceding wherein aspects, the wherein GBA the nucleotide GBA nucleotide

1300 nucleotides of SEQ ID NO: 1.

28. The polynucleotide of any one of the preceding aspects, wherein the GBA nucleotide

1300 nucleotides of SEQ ID NO: 1.

29. Thepolynucleotide 29. The polynucleotideofofany anyone oneofofthe thepreceding precedingaspects, aspects,wherein whereinthe theGBA GBAnucleotide nucleotide

sequence comprises a sequence that is at least 98% identical SEQ ID NO: 1.

30. The polynucleotide of any one of the preceding aspects, wherein the GBA nucleotide

sequence comprises a sequence that is at least 99% identical SEQ ID NO: 1.

31. 31. The polynucleotide The ofof polynucleotide any one any ofof one the preceding the aspects, preceding wherein aspects, the wherein GBA the nucleotide GBA nucleotide

sequence comprises a sequence that is at least 98% identical to SEQ ID NO: 5.

32. Thepolynucleotide 32. The polynucleotideofofany anyone oneofofthe thepreceding precedingaspects, aspects,wherein whereinthe theGBA GBAnucleotide nucleotide

sequence comprises a sequence that is at least 99% identical to SEQ ID NO: 5.

33. The polynucleotide of any one of the preceding aspects, wherein at least a portion of

the GBA nucleotide sequence is codon-optimised.

34. The polynucleotide of aspect 33, wherein the portion of the GBA nucleotide

sequence that is codon-optimised is codon-optimised for expression in human liver

cells.

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35. The polynucleotide of aspect 33, wherein the GBA nucleotide sequence is codon-

optimised for expression in human liver cells.

36. The polynucleotide of any one of aspects 2 to 35, wherein the portion of the GBA

nucleotide sequence that is codon-optimised is a contiguous portion.

37. 37. The polynucleotide The ofof polynucleotide any one any ofof one aspects 2 2 aspects toto 36, wherein 36, the wherein portion the ofof portion the GBA the GBA

nucleotide sequence that is codon-optimised is at least 1000, at least 1200, at least

1300, less than 1494, between 1000 and 1494, between 1300 and 1494, or around

1494 nucleotides in length.

38. 38. The polynucleotide The ofof polynucleotide any one any ofof one aspects 2 2 aspects toto 37, wherein 37, the wherein portion the ofof portion the GBA the GBA

nucleotide sequence that is codon-optimised corresponds to a mature GCase protein.

39. The polynucleotide of any one of aspects 2 to 38, wherein the portion of the GBA

nucleotide sequence that is codon-optimised does not encode all or a portion of a

signal peptide.

40. 40. The polynucleotide The ofof polynucleotide any one any ofof one aspects 2 2 aspects toto 39, wherein 39, the wherein GBA the nucleotide GBA nucleotide

sequence or the portion of the GBA nucleotide sequence that is codon-optimised

comprises a reduced number of CpGs compared to a corresponding portion of a wild

type GBA nucleotide sequence.

41. The polynucleotide of aspect 40, wherein the GBA nucleotide sequence or the

portion of the GBA nucleotide sequence that is codon-optimised comprises less than

40, less than 20, less than 18, less than 10, or less than 5 CpGs.

42. The polynucleotide of aspect 41, wherein the GBA nucleotide sequence or the

5, less than 4, less than 3, or less than 2 CpGs per 100 nucleotides.

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43. 43. The polynucleotide The ofof polynucleotide aspect 4141 aspect oror 42, wherein 42, the wherein GBA the nucleotide GBA sequence nucleotide oror sequence the the

portion of the GBA nucleotide sequence that is codon-optimised is CpG-free.

Thepolynucleotide 44. The polynucleotideofofany anyone oneofofaspects aspects4040toto43, 43,wherein whereinthe thewild wildtype typeGBA GBA

nucleotide sequence is SEQ ID NO: 9.

45. The polynucleotide of any one of aspects 2 to 44, wherein the portion of the GBA

nucleotide sequence that is codon-optimised is at least 80%, at least 85%, at least

90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100%

identical to a fragment of at least 1000, at least 1200, at least 1300, less than 1494,

between 1000 and 1494, between 1300 and 1494, or around 1494 nucleotides of any

one of SEQ ID NO: 1-4.

46. Thepolynucleotide 46. The polynucleotideofofaspect aspect45, 45,wherein whereinthe theportion portionofofthe theGBA GBAnucleotide nucleotide

sequence that is codon-optimised is at least 80%, at least 85%, at least 90%, at least

95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to

any one of SEQ ID NO: 1-4.

47. The polynucleotide of any one of aspects 2 to 46, wherein the portion of the GBA

between 1000 and 1494, between 1300 and 1494, or around 1494 nucleotides of SEQ

ID NO: 1.

48. 48. The polynucleotide of aspect 47, wherein the portion of the GBA nucleotide

SEQ ID NO: 1.

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49. The The polynucleotide polynucleotide of of any any one one of of aspects aspects 22 to to 48, 48, wherein wherein the the portion portion of of the the GBA GBA

nucleotide sequence that is codon-optimised is at least 99.5%, at least 99.8%, or

100% identical to a fragment of at least 1300 nucleotides of SEQ ID NO: 1.

50. 50. The Thepolynucleotide polynucleotideofofaspect aspect49, 49,wherein whereinthe theportion portionofofthe theGBA GBAnucleotide nucleotide

sequence that is codon-optimised is at least 99%, at least 99.5%, at least 99.8%, or

100% identical to SEQ ID NO: 1.

51. The polynucleotide of any one of the preceding aspects, wherein the GBA nucleotide

sequence comprises a portion that is not codon-optimised.

52. 52. The polynucleotide The ofof polynucleotide aspect 51, aspect wherein 51, the wherein portion the that portion isis that not codon-optimised not codon-optimised

encodes all or a portion of a GCase signal peptide.

53. The polynucleotide of aspect 51 or 52, wherein the portion that is not codon-

optimised is at least 80, at least 90, at least 100, at least 110, less than 200, less than

170, less than 140, or around 117 nucleotides.

54. Thepolynucleotide 54. The polynucleotide of ofany anyone of of one aspects 51 to aspects 5153, to wherein the portion 53, wherein that is not the portion that is not

codon-optimised comprises 1 or more CpGs.

55. Thepolynucleotide 55. The polynucleotideofofany anyone oneofofthe thepreceding precedingaspects, aspects,wherein whereinthe thepolynucleotide polynucleotide

further comprises a transcription regulatory element.

56. The polynucleotide of aspect 55, wherein the transcription regulatory element

comprises a liver-specific promoter.

57. 57. The Thepolynucleotide polynucleotideofofaspect aspect5555oror56, 56,wherein whereinthe thetranscription transcriptionregulatory regulatoryelement element

comprises an A1AT promoter or a fragment of an A1AT promoter.

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58. The polynucleotide of aspect 57, wherein the fragment of an A1AT promoter is at

between 150 and 225, between 150 and 300, or between 180 and 255 nucleotides in

length.

59. 59. The Thepolynucleotide polynucleotideofofaspect aspect58, 58,wherein whereinthe thefragment fragmentofofananA1AT A1ATpromoter promoterisis

between 180 and 255 nucleotides in length.

60. The polynucleotide of any one of the preceding aspects, wherein the polynucleotide

comprises a promoter that is at least 80%, at least 85%, at least 90%, at least 95%, at

least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID

NO: 12 or SEQ ID NO: 15.

61. The polynucleotide of aspect 60, wherein the polynucleotide comprises a promoter

that is at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to

SEQ ID NO. 12 or SEQ ID NO: 15.

62. 62. The polynucleotide The ofof polynucleotide aspect 61, aspect wherein 61, the wherein polynucleotide the comprises polynucleotide a a comprises promoter ofof promoter

SEQ ID NO. 12 or SEQ ID NO: 15.

63. 63. The polynucleotide The ofof polynucleotide any one any ofof one aspects 5555 aspects toto 62, wherein 62, the wherein transcription the transcription

regulatory element comprises a fragment of an A1AT promoter that is equal to or

less than 418 nucleotides, equal to or less than 255 nucleotides, or equal to or less

than 185 nucleotides in length and comprises SEQ ID NO: 12.

64. The polynucleotide of any one of aspects 55 to 63, wherein the transcription

regulatory element comprises an enhancer.

65. 65. The polynucleotide The ofof polynucleotide aspect 64, aspect wherein 64, the wherein enhancer the isis enhancer anan HCR enhancer HCR oror enhancer a a

fragment of an HCR enhancer.

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66. The polynucleotide of aspect 65, wherein the fragment of an HCR enhancer is a

fragment of at least 80, at least 90, at least 100, less than 192, between 80 and 192,

between 90 and 192, between 100 and 250, or between 117 and 192 nucleotides in

length.

67. 67. The polynucleotide The ofof polynucleotide aspect 66, aspect wherein 66, the wherein fragment the ofof fragment anan HCR enhancer HCR isis enhancer

between 117 and 192 nucleotides in length.

68. 68. The polynucleotide The ofof polynucleotide any one any ofof one the preceding the aspects, preceding wherein aspects, the wherein polynucleotide the polynucleotide

comprises an enhancer that is at least 80%, at least 85%, at least 90%, at least 95% at

least 98%,atatleast least 98%, least 99%, 99%, at least at least 99.5%, 99.5%, at 99.8%, at least least or 99.8%, or 100% identical 100% identical to SEQ ID to SEQ ID

NO: 11 or SEQ ID NO: 16.

69. The polynucleotide of aspect 68, wherein the polynucleotide comprises an enhancer

SEQ ID NO: 11 or SEQ ID NO: 16.

70. The polynucleotide of aspect 69, wherein the polynucleotide comprises an enhancer

of SEQ ID NO: 11 or SEQ ID NO: 16.

71. 71. The polynucleotide The ofof polynucleotide any one any ofof one aspects 5555 aspects toto 70, wherein 70, the wherein transcription the transcription

regulatory element comprises a fragment of an HCR enhancer that is equal to or less

than 321 nucleotides, equal to or less than 192 nucleotides or equal to or less than

117 nucleotides in length and comprises SEQ ID NO: 11.

72. 72. The polynucleotide The ofof polynucleotide any one any ofof one aspects 5555 aspects toto 71, wherein 71, the wherein transcription the transcription

regulatory element is at least 80%, at least 85%, at least 90%, at least 95%, at least

98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO:

10.

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73. 73. The polynucleotide The ofof polynucleotide aspect 72, aspect wherein 72, the wherein transcription the regulatory transcription element regulatory has element a a has

sequence of SEQ ID NO: 10.

74. The polynucleotide of any one of the preceding aspects, wherein:

(i) (i) the GBA nucleotide sequence comprises a sequence that is at least

SEQ ID NOs: 1 or 5; and

(ii) (ii) the polynucleotide comprises a promoter that is at least 98%, at least

99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO. 12 and/or an

enhancer element that is at least 98%, at least 99%, at least 99.5%, at least 99.8% or

100% identical to SEQ ID NO: 11.

75. The polynucleotide of any one of the preceding aspects, wherein:

(i) (i) the GBA nucleotide sequence comprises a sequence that is at least

SEQ ID NOs: 1 or 5; and

(ii) (ii) the polynucleotide comprises a transcription regulatory element that

is at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to

SEQ ID NO. 10.

76. 76. The polynucleotide The ofof polynucleotide aspect 57, aspect wherein 57, the wherein A1AT the promoter A1AT oror promoter fragment ofof fragment anan

A1AT promoter is at least 200, at least 250, at least 300, less than 500, between 200

and 500, between 250 and 500, between 350 and 450, or around 418 nucleotides in

length.

77. 77. The polynucleotide The ofof polynucleotide aspect 76, aspect wherein 76, the wherein A1AT the promoter A1AT oror promoter fragment ofof fragment anan

A1AT promoter is between 350 and 450 nucleotides in length.

78. The polynucleotide of aspect 65, wherein the HCR enhancer or fragment of an HCR

enhancer is a fragment of at least 150, at least 190, at least 230, less than 400,

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between 150 and 400, between 190 and 370, between 230 and 340, between 250 and

340, or around 321 nucleotides in length.

79. 79. The polynucleotide The ofof polynucleotide aspect 78, aspect wherein 78, the wherein HCR the enhancer HCR oror enhancer fragment ofof fragment anan HCR HCR

enhancer is between 250 and 340 nucleotides in length.

80. 80. The polynucleotide The ofof polynucleotide any one any ofof one aspects 5555 aspects toto 79, wherein 79, the wherein transcription the transcription

14.

81. 81. The polynucleotide The ofof polynucleotide aspect 80, aspect wherein 80, the wherein transcription the regulatory transcription element regulatory has element a a has

sequence of SEQ ID NO: 14.

82. 82. The polynucleotide The ofof polynucleotide any one any ofof one aspects 1 1 aspects toto 5656 oror 7676 toto 79, wherein: 79, wherein:

(i) the GBA nucleotide sequence comprises a sequence that is at least

SEQ ID NOs: 1 or 5; and

(iii) (iii) the polynucleotide comprises a transcription regulatory element that

SEQ ID NO: 14.

83. 83. The polynucleotide The ofof polynucleotide any one any ofof one aspects 1 1 aspects toto 5656 oror 7676 toto 79, wherein: 79, wherein:

(i) the GBA nucleotide sequence comprises a sequence that is at least

SEQ ID NOs: 1 or 5; and

(ii) the polynucleotide comprises a promoter that is at least 98%, at least

99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO. 15 and/or an

100% identical to SEQ ID NO: 16.

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84. The polynucleotide of any one of the preceding aspects, wherein the GCase encoded

by the GBA nucleotide sequence is expressed in human liver cells at higher levels

compared to a GCase encoded by a wild type GBA nucleotide sequence in an

otherwise identical reference polynucleotide.

85. The polynucleotide of any one of the preceding aspects, wherein the GCase encoded

by the GBA nucleotide sequence is expressed in human liver cells at least 1. 1x, at

least 1.2x, at least 1.3x, at least 1.4x, or at least 1.5x higher compared to a GCase

encoded by a wild type GBA nucleotide sequence in a reference polynucleotide.

86. 86. The polynucleotide The ofof polynucleotide aspect 8484 aspect oror 85, wherein 85, the wherein reference the polynucleotide reference polynucleotide

comprises a wild type GBA nucleotide sequence of SEQ ID NO: 9.

87. 87. The polynucleotide The ofof polynucleotide aspect 86, aspect wherein 86, the wherein reference the polynucleotide reference comprises polynucleotide a a comprises

promoter of SEQ ID NO: 13.

88. The polynucleotide of any one of the preceding aspects, wherein the GCase encoded

by the GBA nucleotide sequence is expressed in human liver cells at higher or non-

statistically significant statistically different significant levels different compared levels to GCasetoencoded compared GCase by an otherwise encoded by an otherwise

identical reference polynucleotide comprising a GBA nucleotide sequence of SEQ

ID NO: 9 and operably linked to a promoter of SEQ ID NO: 13.

89. 89. The polynucleotide The ofof polynucleotide any one any ofof one the preceding the aspects, preceding wherein aspects, the wherein polynucleotide the polynucleotide

comprises DNA or RNA, RNA.

90. A viral particle comprising a recombinant genome comprising the polynucleotide of

any one any one of ofthe thepreceding aspects. preceding aspects.

91. TheThe viral particle viral of of particle aspect 90,90, aspect which is is which an an AAV, adenoviral, AAV, or or adenoviral, lentiviral viral lentiviral viral

particle.

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92. The viral particle of aspect 91, which is an AAV viral particle.

93. The viral particle of any one of aspects 90 to 92, wherein the viral particle comprises

a liver-tropic or CNS-tropic capsid.

94. The viral particle of aspect 93, wherein the liver-tropic capsid comprises a sequence

at least 98%, at least 99%, or at least 99.5% identical to a fragment of at least 600, at

least 650, at least 700, between 600 and 736, between 650 and 736, or between 700

and 736 amino acids of SEQ ID NO: 19 or 20.

95. The viral particle of aspect 94, wherein the liver-tropic capsid comprises a sequence

at least 99% identical to SEQ ID NO: 19.

96. The viral particle of aspect 94, wherein the liver-tropic capsid comprises a sequence

at least 99% identical to SEQ ID NO: 20.

97. The viral particle of aspect 93, wherein the CNS-tropic capsid comprises a sequence

least 650, at least 700, between 600 and 736, between 650 and 736 or between 700

and 736 amino acids of SEQ ID NO: 21.

98. Theviral 98. The viralparticle particleofofaspect aspect97, 97,wherein whereinthe theCNS-tropic CNS-tropiccapsid capsidcomprises comprisesa asequence sequence

at least 99% identical to SEQ ID NO: 21.

99. The viral particle of any one of aspects 90 to 98, wherein the recombinant genome

further comprises: further comprises:

a) AAV2 ITRs; b) a poly A sequence; and/or

c) an intron.

100. The viral particle of aspect 99, wherein the recombinant genome is single-stranded.

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101. The viral particle of any one of aspects 90 to 100, wherein on transduction into Huh-

7 cells, the viral particle expresses GCase or a fragment thereof such that the GCase

activity in the transduced cell is greater than the activity of GCase or a fragment

thereof in a cell transduced with an otherwise identical viral particle comprising a

GBA nucleotide sequence of SEQ ID NO: 9.

102. The viral particle of any one of aspects 90 to 101, wherein on transduction into Huh-

activity in the transduced cell is at least 2x, at least 3x, at least 4x, at least 5x, at least

10x, or at least 20x greater than the activity of GCase or a fragment thereof in a cell

transduced with an otherwise identical viral particle comprising a GBA nucleotide

sequence of SEQ ID NO: 9.

103. The viral particle of aspect 101 or 102, wherein the activity is measured using a

fluorometric substrate which is specific for GCase.

104. A composition comprising the polynucleotide or viral particle of any one of the

preceding aspects and a pharmaceutically acceptable excipient.

105. The polynucleotide, viral particle or composition of any one of the preceding aspects

for use in a method of treatment.

106. The polynucleotide, viral particle or composition for use of aspect 105, wherein the

method of treatment comprises administering an effective amount of the

polynucleotide, polynucleotide, composition composition or or viral viral particle particle of of any any one one of of aspects aspects 11 to to 104 104 to to aa

patient.

107. A method of treatment comprising administering an effective amount of the

polynucleotide, composition or viral particle of any one of aspects 1 to 104 to a

patient.

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108. Use of the polynucleotide, viral particle or composition of any one of aspects 1 to

104 in the manufacture of a medicament for use in a method of treatment.

109. The use of aspect 108, wherein the method of treatment comprises administering an

effective amount of the polynucleotide or viral particle of any one of aspects 1 to 104

to a patient.

110. The polynucleotide, viral particle, composition, use or method of any one of aspects

105 to 109, wherein the method of treatment is a method of treating a disease

associated with GCase deficiency.

111. The polynucleotide, viral particle, composition, use or method of any one of aspects

105 to 109, wherein the method of treatment is a method of treating Parkinson's

disease.

112. The polynucleotide, viral particle, composition, use or method of any one of aspects

105 to 109, wherein the method of treatment is a method of treating Gaucher disease.

113. The polynucleotide, viral particle, composition, use or method of aspect 112, wherein

the Gaucher disease is Gaucher disease type I.

114. The polynucleotide, viral particle, composition, use or method of aspect 112, wherein

the Gaucher disease is Gaucher disease type II.

115. 115. The The polynucleotide, polynucleotide, viral viral particle, particle, composition, composition, use use or or method method of of aspect aspect 112, 112, wherein wherein

the Gaucher disease is Gaucher disease type III.

116. The polynucleotide, viral particle, composition, use or method of any one of aspects

112 to 115, wherein the patient has antibodies or inhibitors to a recombinant GCase

with which the patient has previously been treated as part of an enzyme replacement

therapy.

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117. Use of the polynucleotide, viral particle or composition of any one of aspects 1 to

104 in the manufacture of a medicament for achieving a stable GCase activity in a

subject.

118. Use of the polynucleotide, viral particle or composition of any one of aspects 1 to

104 in the manufacture of a medicament for providing greater GCase bioavailability

in a subject compared to the bioavailability from GCase enzyme replacement

therapy, wherein the bioavailability is measured over a period of 2 weeks from

administration.

119. A method of achieving a stable GCase activity in a subject by administering to the

subject the polynucleotide, viral particle or composition of any one of aspects 1 to

104.

120. A method of providing greater GCase bioavailability in a subject compared to the

bioavailability bioavailability from from GCase GCase enzyme enzyme replacement replacement therapy therapy by by administering administering to to the the

104, wherein the bioavailability is measured over a period of 2 weeks from

administration. administration.

121. The method or use of any one of aspects 117 to 120, wherein achieving a stable

GCase activity in a subject or providing greater GCase bioavailability in a subject

treats a disease in the subject.

122. The polynucleotide, viral particle or composition of any one of aspects 1 to 104, for

use in a method of expressing the GBA nucleotide sequence and achieving a stable

GCase activity in a subject.

123. The polynucleotide, viral particle or composition of any one of aspects 1 to 104, for

use in a method of expressing the GBA nucleotide sequence and providing greater

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GCase bioavailability in a subject compared to the bioavailability from GCase

enzyme replacement therapy, wherein the bioavailability is measured over a period

of 2 weeks from administration.

124. The polynucleotide, viral particle or composition for use of aspect 122 or 123,

wherein achieving a stable GCase activity and/or providing greater GCase

bioavailability leads to the treatment of a disease in the subject.

125. The polynucleotide, viral particle or composition for use, use or method of any one

of aspects 117 to 124, wherein the GCase activity and/or bioavailability is measured

using a fluorometric substrate which is specific for GCase.

126. The polynucleotide, viral particle or composition for use, use or method of any one

of aspects 117 to 125, wherein the GCase activity is measured in the serum or plasma

of the subject.

127. The polynucleotide, viral particle or composition for use, use or method of any one

of aspects 117 to 126, wherein the GCase activity is measured in the macrophages of

the subject.

128. The polynucleotide, viral particle or composition for use, use or method of any one

of aspects 117 to 127, wherein the GCase activity is stable at a level of at least 1, at

least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9

umol/h/ml µmol/h/ml in the subject.

129. The polynucleotide, viral particle or composition for use, use or method of any one

of aspects 117 to 128, wherein the GCase activity is stable at a level of at least 3

umol/h/ml µmol/h/ml in the subject.

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130. The polynucleotide, viral particle or composition for use, use or method of any one

of aspects 117 to 129, wherein the GCase activity is stable at a level of at least 5

umol/h/ml µmol/h/ml in the subject.

131. The polynucleotide, viral particle or composition for use, use or method of any one

of aspects 117 to 130, wherein the GCase activity is stable at a level of at least 9

umol/h/ml µmol/h/ml in the subject.

132. The polynucleotide, viral particle or composition for use, use or method of any one

of aspects 117 to 131, wherein the method comprises administering an effective dose

of the polynucleotide, viral particle or composition to the subject.

133. The polynucleotide, viral particle or composition for use, use or method of any one

of aspects 117 to 132, wherein the stable GCase activity is a GCase activity of at

least 10%, at least 20%, at least 30%, at least 40%, or at least 50% relative to the

GCase activity of a healthy subject.

134. The polynucleotide, viral particle or composition for use, use or method of any one

of aspects 117 to 133, wherein the stable GCase activity is a GCase activity of

between 10% and 100%, between 20% and 90%, between 30% and 70%, between

40% and 70%, or between 50% and 70% relative to the GCase activity of a healthy

subject.

135. The polynucleotide, viral particle or composition for use, use or method of any one

of aspects 117 to 134, wherein the stable GCase activity is stable for at least 5 weeks

from administration.

136. The polynucleotide, viral particle or composition for use, use or method of any one

of aspects 117 to 135, wherein the stable GCase activity is stable for at least 10

weeks from administration.

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137. The polynucleotide, viral particle or composition for use, use or method of any one

of aspects 117 to 136, wherein the stable GCase activity is stable for at least 15

weeks from administration.

138. The polynucleotide, viral particle or composition for use, use or method of any one

of aspects 117 to 137, wherein the stable GCase activity is stable for at least 20

weeks from administration.

139. The polynucleotide, viral particle or composition for use, use or method of any one

of aspects 117 to 138, wherein the stable GCase activity is stable for at least 25

weeks from administration.

140. The polynucleotide, viral particle or composition for use, use or method of any one

of aspects 117 to 139, wherein the stable GCase activity is stable for at least 30

weeks from administration.

141. The polynucleotide, viral particle or composition for use, use or method of any one

of aspects 117 to 140, wherein the stable GCase activity is stable for at least 35

weeks from administration.

142. The polynucleotide, viral particle or composition for use, use or method of any one

of aspects 117 to 141, wherein the stable GCase activity is stable for at least 40

weeks after administration.

143. The polynucleotide, viral particle or composition for use, use or method of any one

of aspects 117 to 142, wherein the method achieves a greater GCase activity in the

liver, spleen, and/or bone marrow of the subject at least 5, at least 10, at least 15, at

least 20, at least 25, at least 30, or at least 35 weeks after administration when

compared to the activity measured in a subject administered an effective dose of a

GCase enzyme replacement therapy, when measured in the same assay at the same

time point after administration.

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144. The polynucleotide, viral particle or composition for use, use or method of any one

of aspects 117 to 143, wherein the method achieves a greater GCase bioavailability

in the liver spleen and/or bone marrow subject over a period of at least 5, at least 10,

at least 15, at least 20, at least 25, at least 30, or at least 35 weeks after administration

when compared to the bioavailability measured in a subject administered an effective

dose of a GCase enzyme replacement therapy, when measured in the same assay at

the same time point after administration.

145. The polynucleotide, viral particle or composition for use, use or method of any one

of aspects 118, 120 or 123 to 144, wherein the GCase enzyme replacement therapy

comprises administration of a GCase polypeptide having the sequence of SEQ ID

NO: 25.

146. The polynucleotide, viral particle or composition for use, use or method of aspect

145, wherein the GCase enzyme replacement therapy comprises administration of the

GCase polypeptide at a dose of between 40 and 100, between 50 and 80, between 60

and 70, or around 60 U/kg BW.

147. The polynucleotide, viral particle or composition for use, use or method of any one

of aspects 121 or 124 to 146, wherein the disease is Gaucher disease.

148. The polynucleotide, viral particle or composition for use, use or method of aspect

147, wherein the Gaucher disease is Gaucher disease type I.

149. The polynucleotide, viral particle or composition for use, use or method of aspect

147, wherein the Gaucher disease is Gaucher disease type II.

150. The polynucleotide, viral particle or composition for use, use or method of aspect

147, wherein the Gaucher disease is Gaucher disease type III.

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151. The polynucleotide, viral particle or composition for use, use or method of any one

of aspects 117 to 150, wherein the patient has antibodies or inhibitors to a

recombinant GCase with which the patient has previously been treated as part of an

enzyme replacement therapy.

152. Use of the polynucleotide, viral particle or composition of any one of aspects 1 to

104 in the manufacture of a medicament for reducing the levels of hexosylceramide

and/or hexosylsphingosine in a subject suffering from a disease or condition

associated with GCase deficiency.

153. A method of reducing the levels of hexosylceramide and/or hexosylsphingosine in a

subject suffering from a disease or condition associated with GCase deficiency by

administering to the subject the polynucleotide, viral particle or composition of any

one of aspects 1 to 104.

154. The use or method of aspect 152 or 153 wherein reducing the levels of

hexosylceramide and/or hexosylsphingosine in a subject treats the disease or

condition associated with GCase deficiency.

155. The polynucleotide, viral particle or composition of any one of aspects 1 to 104, for

use in a method of reducing hexosylceramide and/or hexosylsphingosine levels in a

subject suffering from a disease or condition associated with GCase deficiency,

optionally wherein reducing hexosylceramide and/or hexosylsphingosine levels leads

to the treatment of the disease or condition associated with GCase deficiency.

156. The polynucleotide, viral particle or composition for use, use or method of any one

of aspects 152 to 155, wherein the hexosylceramide and/or hexosylsphingosine levels

are reduced by 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6

times or more, 2 to 3 times, 2 to 4 times, 2 to 5 times, 2 to 6 times, or 3 to 5 times

when compared to the hexosylceramide and/or hexosylsphingosine levels at the time

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of administration of the polynucleotide, viral particle or composition of any one of

aspects 1 to 104.

157. The polynucleotide, viral particle or composition for use, use or method of any one

of aspects 152 to 156, wherein the reduction in hexosylceramide and/or

hexosylsphingosine levels is greater than the reduction achieved in a subject

administered an effective dose of a GCase enzyme replacement therapy, optionally

when the hexosylceramide and/or hexosylsphingosine levels are measured at least 6

weeks, at least 8 weeks, at least 10 weeks or at least 12 weeks after administration.

158. The polynucleotide, viral particle or composition for use, use or method of aspect

157, wherein the GCase enzyme replacement therapy comprises administration of a

GCase polypeptide having the sequence of SEQ ID NO: 25.

159. The polynucleotide, viral particle or composition for use, use or method of aspect

158, wherein the GCase enzyme replacement therapy comprises administration of the

and 70, or around 60 U/kg BW.

160. The polynucleotide, viral particle or composition for use, use or method of any one

of aspects 152 to 159, wherein the hexosylceramide and/or hexosylsphingosine levels

are measured in the macrophages of the subject.

161. The polynucleotide, viral particle or composition for use, use or method of any one

of aspects 152 to 160, wherein the hexosylceramide and/or hexosylsphingosine levels

are measured in the spleen of the subject.

162. The polynucleotide, viral particle or composition for use, use or method of any one

of aspects 152 to 161, wherein the hexosylceramide and/or hexosylsphingosine levels

are measured in the liver of the subject.

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163. The polynucleotide, viral particle or composition for use, use or method of any one

of aspects 152 to 162, wherein the hexosylceramide and/or hexosylsphingosine levels

are measured in the serum of the subject.

164. The polynucleotide, viral particle or composition for use, use or method of any one

of aspects 152 to 163, wherein the hexosylceramide and/or hexosylsphingosine levels

are measured by mass spectrometry.

165 The polynucleotide, viral particle or composition for use, use or method of any one

of aspects 152 to 164, wherein the disease is Gaucher disease.

166. The polynucleotide, viral particle or composition for use, use or method of aspect

165, wherein the Gaucher disease is Gaucher disease type I.

167. The polynucleotide, viral particle or composition for use, use or method of aspect

165, wherein the Gaucher disease is Gaucher disease type II.

168. The polynucleotide, viral particle or composition for use, use or method of aspect

165, wherein the Gaucher disease is Gaucher disease type III.

169. The polynucleotide, viral particle or composition for use, use or method of any one

of aspects 152 to 168, wherein the patient has antibodies or inhibitors to a

enzyme replacement therapy.

PCT/GB2020/050251

Further aspects of the invention

The invention is also described in the following aspects.

1. 1. A polynucleotide comprising a GBA nucleotide sequence, wherein the GBA

type, optionally wherein the portion of the GBA nucleotide sequence that is not wild

type is codon-optimised, more optionally wherein the GBA nucleotide sequence

encodes a GCase protein or a fragment thereof and comprises a sequence that is at

least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at

least 99.8%, or 100% identical to a nucleotide sequence of any one of SEQ ID NO:

1-8. 1-8.

2. 2. A polynucleotide comprising a GBA nucleotide sequence, wherein the GBA

nucleotide sequence encodes a GCase protein or a fragment thereof and comprises a a sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at

1000 and 1600, between 1300 and 1494, between 1300 and 1611, around 1494, or

around 1611 nucleotides of any one of SEQ ID NO: 1-8.

3. The polynucleotide of any one of aspects 1 to 2, wherein at least a portion of the

GBA nucleotide sequence is codon-optimised.

4. 4. The polynucleotide of aspect 3, wherein:

(a) the at least a portion of the GBA nucleotide sequence that is codon-

optimised is codon-optimised for expression in human liver cells;

(b) the portion of the GBA nucleotide sequence that is codon-optimised is a

contiguous portion;

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(c) the portion of the GBA nucleotide sequence that is codon-optimised is at

least 1000, at least 1200, at least 1300, less than 1494, between 1000 and 1494,

between 1300 and 1494, or around 1494 nucleotides in length;

(d) the portion of the GBA nucleotide sequence that is codon-optimised

corresponds to a mature GCase protein;

(e) the portion of the GBA nucleotide sequence that is codon-optimised does

not encode all or a portion of a signal peptide;

(f) (f) the GBA nucleotide sequence or the portion of the GBA nucleotide

sequence that is codon-optimised comprises a reduced number of CpGs compared to

a corresponding portion of a wild type GBA nucleotide sequence; optionally wherein

the GBA nucleotide sequence or the portion of the GBA nucleotide sequence that is

codon-optimised comprises less than 40, less than 20, less than 18, less than 10, or

less than 5 CpGs, more optionally wherein the GBA nucleotide sequence or the

5, less than 4, less than 3, or less than 2 CpGs per 100 nucleotides, more optionally

wherein the GBA nucleotide sequence or the portion of the GBA nucleotide

sequence that is codon-optimised is CpG-free, preferably wherein the wild type GBA

nucleotide sequence is SEQ ID NO: 9; and/or

(g) the portion of the GBA nucleotide sequence that is codon-optimised is at

least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least

99.5%, at least 99.8%, or 100% identical to a fragment of at least 1000, at least 1200,

at least 1300, less than 1494, between 1000 and 1494, between 1300 and 1494, or

around 1494 nucleotides of any one of SEQ ID NO: 1-4.

5. The polynucleotide of any one of the preceding aspects, wherein the GBA nucleotide

sequence comprises a portion that is not codon-optimised, optionally wherein:

(a) the portion that is not codon-optimised encodes all or a portion of a GCase

signal peptide;

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(b) the the portion portion that that is is not not codon-optimised codon-optimised is is at at least least 80, 80, at at least least 90, 90, at at least least 100, 100,

at least 110, less than 200, less than 170, less than 140, or around 117 nucleotides;

and/or

(c) the portion that is not codon-optimised comprises 1 or more CpGs.

6. The polynucleotide of any one of the preceding aspects, wherein the polynucleotide

further comprises a transcription regulatory element, optionally wherein the

transcription regulatory element comprises a liver-specific promoter and/or an

enhancer.

7. 7. The polynucleotide of aspect 6, wherein the transcription regulatory element

comprises an A1AT promoter or a fragment of an A1AT promoter, optionally

wherein (a) (a) the A1AT promoter or the fragment of an A1AT promoter is at least 100, at

least 120, at least 150, at least 180, less than 255, between 100 and 255, between 150

and 225, between 150 and 300, or between 180 and 255 nucleotides in length, more

optionally wherein the fragment of an A1AT promoter is between 180 and 255

nucleotides in length;

(b) (b) the A1AT promoter or the fragment of an A1AT promoter is at least 200, at

least 250, at least 300, less than 500, between 200 and 500, between 250 and 500,

between 350 and 450, or around 418 nucleotides in length, more optionally wherein

the fragment of an A1AT promoter is between 350 and 450 nucleotides in length;

(c) the polynucleotide comprises a promoter that is at least 80%, at least 85%,

at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%,

or 100% identical to SEQ ID NO: 12 or SEQ ID NO: 15.

8. The polynucleotide of aspects 6 or 7, wherein the enhancer is an HCR enhancer or a

fragment of an HCR enhancer, optionally wherein:

(a) (a) the HCR enhancer or the fragment of an HCR enhancer is a fragment of at

least 80, at least 90, at least 100, less than 192, between 80 and 192, between 90 and

192, between 100 and 250, or between 117 and 192 nucleotides in length, more

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optionally wherein the fragment of an HCR enhancer is between 117 and 192

nucleotides in length;

(b) the HCR enhancer or the fragment of an HCR enhancer is a fragment of at

least 150, at least 190, at least 230, less than 400, between 150 and 400, between 190

and 370, between 230 and 340, between 250 and 340, or around 321 nucleotides in

length, more optionally wherein the fragment of an HCR enhancer is between 250

and 340 nucleotides in length

(c) the polynucleotide comprises an enhancer that is at least 80%, at least 85%,

at least 90%, at least 95% at least 98%, at least 99%, at least 99.5%, at least 99.8%,

or 100% identical to SEQ ID NO: 11 or SEQ ID NO: 16.

9. The polynucleotide of aspect 6, wherein the transcription regulatory element is at

99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 10 or 14.

10. The polynucleotide of any one of the preceding aspects, wherein the GCase encoded

compared to a GCase encoded by a wild type GBA nucleotide sequence in an

otherwise identical reference polynucleotide, optionally wherein the GCase encoded

by the GBA nucleotide sequence is expressed in human liver cells at least 1.1x, at

encoded by a wild type GBA nucleotide sequence in an otherwise identical reference

polynucleotide, more optionally wherein the reference polynucleotide comprises a

wild type GBA nucleotide sequence of SEQ ID NO: 9, optionally wherein the

reference polynucleotide comprises a promoter of SEQ ID NO: 13.

11. 11. A A viral viralparticle particlecomprising comprisinga arecombinant recombinantgenome genomecomprising comprisingthe thepolynucleotide polynucleotideofof

any one any one of ofthe thepreceding aspects. preceding aspects.

12. TheThe viral particle viral of of particle aspect 11,11, aspect which is is which an an AAV, adenoviral, AAV, or or adenoviral, lentiviral viral lentiviral viral

particle, optionally which is an AAV viral particle.

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13. TheThe viral particle viral of of particle anyany oneone of of aspects 11-12, aspects wherein 11-12, thethe wherein viral particle viral comprises particle a a comprises

liver-tropic or CNS-tropic capsid.

14. The viral particle of aspect 13, wherein the liver-tropic capsid comprises a sequence

at least 98%, at least 99%, at least 99.5% to a fragment of at least 600, at least 650,

at least 700, between 600 and 736, between 650 and 736 or between 700 and 736

amino acids of SEQ ID NO: 19, 20 or 24.

15. 15. The viral particle of aspect 13, wherein the CNS-tropic capsid comprises a sequence

at least 700, between 600 and 736, between 650 and 736 or between 700 and 736

amino acids of SEQ ID NO: 21.

16. The viral particle of any one of aspects 11 to 15, wherein the recombinant genome

further comprises:

a) AAV2 ITRs; b) a poly A sequence; and/or

c) an intron;

optionally wherein the recombinant genome is single-stranded.

17. 17. The Theviral viral particle ofany particle of anyone one of of aspects aspects 1116, 11 to to wherein 16, wherein on transduction on transduction into Huh-7 into Huh-7

cells, the viral particle expresses GCase or a fragment thereof such that the GCase

activity activity in in the the transduced transduced cell cell is is greater greater than than the the activity activity of of GCase GCase or or a a fragment fragment

GBA nucleotide sequence of SEQ ID NO: 9, optionally wherein on transduction into

Huh-7 cells, the viral particle expresses GCase or a fragment thereof such that the

GCase activity in the transduced cell is at least 2x, at least 3x, at least 4x, at least 5x,

at least 10x, or at least 20x greater than the activity of GCase or a fragment thereof in

a cell transduced with an otherwise identical viral particle comprising a GBA

nucleotide sequence of SEQ ID NO: 9, more optionally wherein the activity is

measured using a fluorometric substrate which is specific for GCase.

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18. A composition comprising the polynucleotide or viral particle of any one of the

preceding aspects and a pharmaceutically acceptable excipient.

19. The polynucleotide, viral particle or composition of any one of the preceding aspects

for use in a method of treatment.

20. 20. The Thepolynucleotide, viralparticle polynucleotide, viral particle or or composition composition forofuse for use of aspect aspect 19, the 19, wherein wherein the

method of treatment comprises administering an effective amount of the

polynucleotide, composition or viral particle of any one of aspects 1 to 17 to a

patient.

21. The polynucleotide, viral particle, or composition for use of any one of aspects 19 to

20, wherein 20, whereinthe method the of treatment method is a method of treatment of treating is a method a diseasea associated of treating with disease associated with

GCase deficiency.

Thepolynucleotide, 22. The 22. polynucleotide, viral viralparticle, or composition particle, for use or composition forofuse any of oneany of aspects one of 19 to aspects 19 to

20, wherein the method of treatment is a method of treating Parkinson's disease.

23. The polynucleotide, viral particle, or composition for use of any one of aspects 19 to

20, wherein the method of treatment is a method of treating Gaucher disease.

24. The polynucleotide, viral particle, or composition for use of aspect 23, wherein the

Gaucher disease is Gaucher disease type I, II or III.

25. Thepolynucleotide, 25. The polynucleotide, viral viralparticle, or composition particle, for use or composition forofuse any of oneany of aspects one of 23 to aspects 23 to

24, wherein the patient has antibodies or inhibitors to a recombinant GCase with

which the patient has previously been treated as part of an enzyme replacement

therapy.

PCT/GB2020/050251

Examples

Example 1 - Methods

Unless specified otherwise, the following general methods were followed in the examples

described below.

rAAV production

AAV2/8 particles were produced by transient transfection of HEK293T cells with plasmids

encoding the AAV Rep and Cap, and adenoviral helper functions, as well the recombinant

genome containing the GBA construct. AAV2/8 particles were purified by aPOROS

CaptureSelect affinity column and were titered by qPCR and characterised by alkaline gel

analysis.

Mouse study design

AAV viral particles carrying the GBA transgene under transcriptional control of the

hepatocyte-specific promoters were administered into the tail vein of wild type (C57BL/6)

male mice at age of 6-8 weeks. AAV dose ranged from 6x1011 6x 10¹¹vg/kg vg/kgto to6x1012 vg/kg, 6x 10¹² as as vg/kg,

herein for each study. For each experiment, an additional group of animals was left

untreated to serve as a control for the effects of treatment. To assess the kinetics and

durability of transgene expression, serum GCase levels were measured at various time

intervals intervals(4-, 8-,8-, (4-, andand 12- 12- - weeks) post weeks) injection. post Mice were injection. Mice followed up to 12up were followed weeks postweeks post to 12

AAV treatment and sacrificed for biochemical and pathological analysis.

Serum and tissue GBA activity assay

B-Glucocerebrosidase (acidß-glucosidase; -Glucocerebrosidase (acid B-glucosidase;GCase) GCase)activity activitywas wasdetermined determined

fluorometrically with 4-Methylumbelliferyl-B-D-glucopyranoside 4-Methylumbelliferyl-ß-D-glucopyranoside (4MU-Glc) as a

substrate. Serum samples were obtained from mouse blood and stored at -80 °C. Tissues

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(liver, spleen, bone marrow) were harvested and snap frozen and lysed. - ß-

Glucocerebrosidase (acid B-glucosidase, ß-glucosidase, GCase) activity was determined fluorometrically

with 4-Methylumbelliferyl-B-D-glucopyranoside 4-Methylumbelliferyl-ß-D-glucopyranoside (4MU-Glc) as a substrate. On the day of

the assay, serum was diluted (0.5 uL, µL, 1:50) and assayed in 50 mM Sodium Citrate, 25 mM

Taurocholate, pH=5.75, 6 mM 4MU-Glc, for 30 min at 37°C. For tissue samples, the

tissue protein lysate was assayed directly. The reaction was stopped by adding one volume

(100 ul) µ1) of stop solution (0.5 M Glycine, 0.3 M NaOH, pH 10.0). Relative fluorescence

levels (RFU) were evaluated with a Spectramax I3X (Molecular devices) using excitation

and emission wavelengths of 365 nm and 445 nm, respectively. Fluorescence levels were

then converted to nanomoles/h/mL (serum) or nmol/h/mg of total protein (tissue) based on

a 4-Methylumbelliferone (4-MU, Sigma-Aldrich) standard curve.

Vector genome copy number

To determine the number of vector genomes per liver cell post- rAAV injection, DNA was

isolated from frozen liver samples using QIAGEN DNeasy Blood and Tissue Kit

(QIAGEN) following manufacturers' instructions. Following DNA isolation, qPCR was

performed using primer sets which bind to a region common to both LSP-S and LSP-L

promoters, allowing estimation of AAV copy number.

Immunohistochemistry

Rabbit anti-human GCase (Abcam ab125065; ab 125065;1:100) 1:100)was wasused usedto tovisualize visualizeGCase GCasein in

mouse tissue. The rat anti-F4/80 (Abcam ab6640; 1:100) was used to visualize mouse

macrophages. The formalin-fixed mouse tissues were deparaffinized with xylene and

ethanol washes, followed by antigen retrieval according to Ventana CC1 product use

recommendations. Immunohistochemistry staining was performed using the Ventana

Discovery XT instrument, using the Ventana DAB Map detection Kit (760-124). Sections

were counterstained with haematoxylin. FITC- and Texas red-conjugated secondary

antibodies were used during immunofluorescent staining. DAPI was used to visualize

nuclei. The signals were visualized by confocal fluorescence microscopy (Zeiss).

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Huh-7 transfection and potency assay

The day before transfection, the liver hepatocyte cell line Huh-7 was plated in a 12 well

plate at the cell density of 3x105 3x 10 cells per well. For transfection, FuGENE was used at the

ratio of 4 ul µl per ug µg of plasmid and added overnight to Huh-7 cells in the presence of 10%

of serum (foetal bovine serum, FBS). Transfection medium was changed, and cells were

incubated for 24 hours with medium supplemented with Insulin-Transferrin-Selenium

(ITS, ThermoFisher Scientific) and 25 mM Hepes buffer. Huh-7 cell transduction, was

performed performedatatdefined multiplicity defined of infection multiplicity (MOI) in of infection the presence (MOI) of serum for in the presence of 24 hours, serum for 24 hours,

followed by a medium change and incubation for 24 hours in fresh medium. 20 ul µl of

medium was used to measure GCase activity using 4MU-Glc as a substrate, as described

above.

Statistical analysis

Statistical analysis was performed using Prism 7 (Graph Pad) software. Columns analysis

was performed by one-way ANOVA. P-values and sample size are indicated in Figure

descriptions.

To approximate bioavailability (AUC), a one-phase decay model equation: Y= (YO (Y0 -

Plateau)*exp(-K*X) + Plateau was used in GraphPad Prism. Y0 is the Y-value when X

(time) is zero and it is expressed in the same units as Y. Plateau is the Y-value at infinite

times, expressed in the same units as Y. K is the rate constant, expressed in reciprocal of

the X-axis time units (i.e. if X is in minutes, then K is expressed in minutes-1. minutes¹). Tau is the

time constant, expressed in the same units as the X-axis and is computed as the reciprocal

of K. Half-life is in the time units of the X-axis, computed as In(2)/K. Span is the

difference between Y0 and Plateau, expressed in the same units as the Y-values. The linear

trapezoidal method was used for the AUC calculation. AUC is expressed as U*h/L where

one unit is defined as the amount of enzyme required to hydrolyse 1 umol/h µmol/h of 4-

-D-glucopyranoside methylumbelliferyl- ß substrate -D-glucopyranoside atat substrate 37°C. 37°C.

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Example 2 - GBA constructs

In order to evaluate if a liver-directed gene therapy approach could be used to treat

Gaucher disease (GD), the human full-length GBA coding sequence (as found in GenBank

accession no. NM_000157.3; SEQ ID NO: 9) was cloned into a liver-specific promoter-

driven adeno-associated virus (AAV) vector. In FLF-PL01 AAV construct (Fig 1A) the

GBA wild type sequence (GBAwt, non-codon-optimized non-codon-optimized)is isdriven drivenby bya aliver-specific liver-specific

promoter referred to herein as 'LSP-S' (SEQ ID NO: 10). In order to determine a

sequence optimal for expression, sequences were designed using a number of different

codon optimisation strategies. In one example AAV construct (FLF-PL28) the GBA

codon sequence was optimized and is driven by the same liver specific promoter LSP-S

(Fig 1B). The FLF-PL64 construct contains the same GBA codon-optimized sequence as

FLF-PL28 but differs in containing a longer transcription regulatory element, referred to

here as 'LSP-L' (SEQ ID NO: 14) instead of LSP-S (Fig 1C).

Example 3 - Analysis of wild type GBA transgene expression

In order to evaluate if the (wild type) GBA construct FLF-PL01 could lead to liver

expression and subsequent secretion of B-glucocerebrosidase ß-glucocerebrosidase (GCase) into the

bloodstream, FLF-PL01 was pseudotyped into AAV2/8. rAAV particles were produced

and titered as described above, and characterised by alkaline gel analysis, prior to be used

in mice. Eight-week-old wild type (C57BL/6) mice were treated with a single injection of

AAV2/8-FLF-PL01 at a dose ranging from 6x1011 6x 10¹¹to to6x 1012 vg/kg. Control (naive) 6x10¹² (naïve) mice

were left untreated. Serum samples were collected at four, eight, and 12 weeks post-AAV

injection and used to evaluate levels of circulating active GCase. GCase activity was

determined and immunohistochemistry staining was performed as described above.

Sections were counterstained with haematoxylin.

Injection of wild-type mice with AAV2/8-FLF-PL01 resulted in an increase in expression

of human GCase in the liver of treated animals (Fig 2A). An increased level of GCase

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expression in liver could be observed with increased vector dose, with around 12-fold

increase increaseobserved observedat at the the vector dose dose vector of 6x of 1011 6xvg/kg 10¹¹ group, vg/kg 43-fold group, at the dose 43-fold atofthe 2x1012 dose of 10¹²

vg/kg, and 57-fold at the dose of 6x1012 6x 10¹²vg/kg vg/kg(Fig (Fig2B). 2B).This Thisdata datashows showsthat thatAAV2/8- AAV2/8-

FLF-PL01 drove expression of GCase to levels that result in significant release of GCase

to the bloodstream and possible access to macrophages in GD affected tissues.

Example 4 - Analysis of in vitro GCase expression from codon-optimised constructs

Codon usage tables of various liver expressed sequences were used in order to generate

GBA sequences codon-optimised throughout the stretch corresponding to the mature

GCase protein (but not the signal peptide-encoding region). With the exception of one such

codon-optimised GBA sequence ('FLF-PL36') the resulting sequences were then further

manually altered to remove CpGs, cryptic splice sites, premature stop codons and

unwanted amino acid substitutions. Twenty-one codon-optimised GBA sequences were

created and tested for GCase expression levels upon transfection in the human liver cell

line line Huh-7. Huh-7.Huh-7 cells Huh-7 werewere cells plated onto aonto plated 12 well a 12plate wellatplate the cell density at the cellofdensity 3x105 per of 3x10 per

well and transfected as described above. Twenty microliters of medium was used to

measure GCase activity using 4MU-Glc as a substrate. Results from this analysis allowed

the identification of GBA codon-optimizations (FLF-PL21, -PL28, -PL30, and -PL36) that

demonstrated increased expression of GCase (relative to wild type GBA sequence, FLF-

PL01) when transfected in Huh-7 cells (Fig 3).

Example 5 - Analysis of in vivo GCase activity from codon-optimised constructs

The four constructs (FLF-PL21, FLF-PL28, FLF-PL30 and FLF-PL36) identified in

example 4 were pseudotyped as AAV2/8 and injection into wild-type mice at the dose of

2x1012 2x10¹² vg/kg. Also included in the experiment was the non-codon-optimised construct

FLF-PL01, as well as a construct (FLF-PL37) containing the same wild type GBA

sequence as FLF-PL01 driven by the strong synthetic promoter CAG. Control (naive) (naïve) mice

were left untreated. At time points up to 36 weeks after injection, animals were sacrificed,

and serum and tissues samples were collected.

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Fig 4A shows the results at 8 weeks post-injection of GCase activity found in mice injected

either with the non-codon optimized GBA sequence driven by the LSP-S promoter (FLF-

PL01), the codon-optimized GBA constructs (FLF-PL21, FLF-PL28, FLF-PL30 and FLF-

PL36) also driven by the LSP-S promoter, and the GBA non-codon-optimized sequence

driven by CAG promoter (FLF-PL37). All four GBA codon-optimized constructs showed

increased levels of GCase activity present in the bloodstream when injected in mice (Fig

4A), relative to FLF-PL01. The FLF-PL28 construct exhibited the greatest increase (about

6-fold) in GCase release to the bloodstream compared to the non-codon-optimised

construct driven by the same LSP-S promoter (FLF-PL01). The elevated level of GCase

driven by FLF-PL28 relative to FLF-PL01 was observed throughout the 36-week study

period (Fig 4B).

Of particular note, the levels of GCase observed in liver-specific promoter-containing FLF-

PL28-injected mice were as high as the GCase levels driven by the FLF-PL37 construct in

which wild type GBA sequence is expressed from the ubiquitous and strong CAG

promoter (Fig 4A).

At end-stage, spleen and bone marrow were collected and fixed in formalin, followed by

paraffin embedding. GBA immunostaining analysis performed on paraffin sections shows

that, in agreement with the circulating GCase levels, tissue uptake of GCase is increased in

mice treated with the FLF-PL28 GBA codon-optimised construct compared to non-codon-

optimised construct FLF-PL01 (Fig 5).

In order to evaluate levels of macrophage uptake in spleen upon liver-directed GBA

expression by FLF-PL28, immunofluorescence analysis with the mouse pan-macrophage

marker F4/80 and a GBA antibody was performed. The majority of F4/80 positive cells

display expression of the human-specific GBA, suggesting that the majority of GCase

uptake in spleen occurs in macrophages (Fig 6).

Example 6 - Analysis of promoter effect on in vivo GCase activity

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To test if promoter engineering could further increase expression from a GBA codon-

optimised sequence, the GBA construct from FLF-PL28 was placed under a liver-specific

promoter (referred to herein as 'LSP-L'; SEQ ID NO: 14) to generate construct FLF-PL64

(Example 2, Fig 1C).

AAV2/8 vectors were prepared with the new construct and injected into wild type mice at

2x10¹² vg/kg. Control (naive) the dose of 2x1012 (naïve) mice were left untreated. After 5 weeks, animals

were sacrificed, and serum and tissues were collected.

GCase activity analysis in serum shows that AAV2/8-FLF-PL64 results in an increased

expression (about 2.5-fold, P=0.0001, one-way ANOVA) of GCase in the mouse

bloodstream compared to mice treated with AAV2/8-FLF-PL28 (Fig 7).

Like construct FLF-PL28, FLF-PL64 allows robust uptake of GCase into GD target tissues

such spleen, bone marrow and lung (Fig 8).

Example 7 - Liver expression selectivity from AAV vectors with GBA constructs

To analyse the selectivity of the LSP-L promoter for a hepatic cell line, eight human-

derived cell lines from a variety of tissues were selected. Details of each cell line and its

origin are summarized in the table below.

Table 1. Human-derived cell lines evaluated in this example

Cell line Origin Growth Species of

origin

HUH-7 Hepatocellular carcinoma (Liver) Adherent Human HUH-7 HEK293T Kidney Adherent Human PANC-1 Pancreas (epithelioid carcinoma) Adherent Human BxPC-3 Pancreas (adenocarcinoma) Adherent Human

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Breast (epithelial; Adherent MCF7 Human adenocarcinoma)

1643 Neuroblastoma Adherent Human Normal lung fibroblast (embryo) Adherent Human MRC-9 697 B-cell B-cell leukaemia leukaemia(early B-cell) (early B-cell) Suspension Suspension Human

The eight human-derived cell lines as described above in Table 1 were grown in either

DMEM, IMDM or RPMI media, supplemented with 10% FBS. For each cell line, 2x104 2x10

cells/well were transduced at a multiplicity of infection (MOI) of 1x105 vg/cellwith 1x10 vg/cell withAAV- AAV-

FLF-PL64 (AAV with liver tropic capsid = SEQ ID NO: 20). All experiments were

performed in duplicate. Cells in suspension were counted and transduced in serum-free

media (300ul/well) (300µl/well) into 48-well plates. For the adherent cell lines, media was aspirated,

followed by washing with PBS (1X) and treatment with 5ml of TripLE for five minutes at

37°C, 5% CO2, to dissociate CO, to dissociate the the cells. cells. The The reaction reaction was was stopped stopped by by adding adding 5ml 5ml of of

complete media. Dissociated cells were counted using a CountessTM Countess IIII Automated Automated Cell Cell

Counter (ThermoFisher) and centrifuged (250 X for five g for minutes), five followed minutes), by by followed

resuspension resuspensioninin complete media complete at a at media density of 2x105 a density of cells/ml. These cells 2x10 cells/ml. were These plated cells intoplated into were

96-well plates (2x104 cells/well) to (2x10 cells/well) to adhere adhere for for five five hours hours prior prior to to transduction. transduction.

Transduction mix was prepared in X-VIVO media (50 ul/well) µl/well) and added to the cells.

After three hours, 100ul/well 100µl/well of complete media was added. One day post-transduction, the

media for each cell line was changed to complete media (+25mM HEPES for secretion

analysis).

GCase activity was determined fluorometrically with 4-Methylumbelliferyl-B-D- 4-Methylumbellifery1-B-D-

glucopyranoside (4MU-Glc) as substrate.

GCase activity was measured from the culture supernatant for each cell line to determine

the levels of GCase secreted following transduction with AAV-FLF-PL64 (Figure 10).

When the LSP-L promoter drives the GBA transgene, GCase secretion was detected in the

HUH-7 cell line alone. The level of active GCase observed in HUH-7 cells was

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approximately 5.0 nmol/h/ml [5.1 0.1 nmol/h/ml]. ± 0.1 No No nmol/h/ml]. detectable levels detectable of of levels active GCase active GCase

were observed for any of the other cell lines analysed.

Example 8 - Comparison with ERT therapy

The goal of this example was to compare FLF-PL64 with VPRIV R (60 U/kg BW) when

administered in mice as a single injection. VPRIV® contains the same amino acid

sequence and a similar glycosylation pattern as the native enzyme, GCase (i.e. SEQ ID

NO: 25), and therefore provides a suitable comparison. Patients undergoing enzyme

replacement therapy (ERT) would be typically treated with an IV infusion of ERT

(duration of infusion of 1-2 hours, clinical dose of VPRIV® is 60 U/kg) on alternate

weeks.

VPRIV R powder (400 Units, Shire), for preparation of a solution for infusion, was

obtained and maintained under refrigeration and protection from light until reconstitution.

One vial (400U) was reconstituted with 4.3 ml of sterile water to achieve a solution at 100

U/ml, as recommended by the manufacturer. Following reconstitution, VPRIV solution

was promptly snap frozen as single-use aliquots and stored at (-80 °C) for later use.

A single IV injection of either VPRIV R (60 U/kg BW) or FLF-PL64 (formulated as

AAV2/8 particles, 2x1012 2x10¹² vg/kg), was administered to wild type mice. Levels of active

GCase in serum and tissue were determined at various time points for up to one week and

also at three weeks and five weeks post-injection. The levels of active GCase were

determined fluorometrically with 4-Methylumbelliferyl-B-D-glucopyranoside (4MU-Glc).

As shown in Figure 11(A), VPRIV is rapidly cleared from murine blood. VPRIV reached a

Cmax of 12.7 umol/h/ml µmol/h/ml at two minutes post-injection; with an estimated half-life of

approximately 5.6 minutes. At approximately 20 minutes post-injection, only residual

levels of active GCase could be detected in serum. These levels remained close to

untreated controls for the remainder of the study period. A comparison of VPRIV with

FLF-PL64 was made by analysing mice with stable expression of GCase (Figure 11B).

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Treatment with FLF-PL64 also led to increased levels of active GCase in murine blood

(Cmax 9.4 umol/h/ml) µmol/h/ml) (Figure 11B). However, although levels of active GCase were not

as high as observed post-VPRIV R injection, these levels remained constant for the

duration of the study. Table 2 below shows the predicted bioavailability during a 2-week

interval in mice following injection of either ERT or FLF-PL64.

Table 2. Predicted bioavailability (AUC) during a 2-week interval in C57BL/6 mice

upon single injection of ERT (60 U/kg BW) or AAV-FLF-PL64 (2x1012 (2x10¹² vg/kg) vg/kg).

ERT (VPRIV®) ERT (VPRIV FLF-PL64 AUC2week (min.U/ml) 18.6 + ± 5.6 3,161.6 + ± 348.2

Figure 12 shows GCase immunostaining in murine liver, spleen and bone marrow

following administration of VPRIV R or AAV2/8-FLF-PL64. A representative image for

each animal group is shown. DAB (3,3'-Diaminobenzidine) was used to visualise GCase

and haematoxylin was used as counterstain. FLF-PL64 treated samples were obtained at

five weeks post-injection, while VPRIV R treated samples were collected as labelled. A

semi-quantitative analysis of the images are represented in Table 3 below:

Table 3: Relative levels of GCase immunoreactivity observed in murine liver, spleen

and bone marrow post-administration of either ERT (VPRIV R) ®) or FLF-PL64. "_"

refers to negative staining for GCase; "+" represents positive staining for GCase.

Group Liver Spleen Bone Marrow Naive Naïve - I - I --

ERT - 20 min + + ++ ++ ++ ERT ERT -- 60 60 min min + +++ ++ ERT - 240 min -/+ + --

ERT - 1440 min -/+ - --

FLF-PL64 +++ +++ ++/+++

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Example 9 - in vivo study of therapeutic potential

1. 1. Methods Methods

Mouse methods

9V/null mice carrying the Gbal mutation D409V/D409V (9V/9V) were used as the

Gaucher disease model in this study. 9V/null mice have a nearly normal lifespan with

visceral abnormalities (inflammation and storage cells) and substrate accumulation (Xu et

al. Am J Pathol. 2003 Nov;163(5):2093-101; Xu et al. PLoS One. 2010 May

20;5(5):e10750). 9V/null mice were generated by crossing mice carrying Gbal mutation

D409V/D409V (9V/9V) and Gbal null/WT. There are approximately two 9V/null

produced in each litter. The strain background of 9V/null and WT mice are C57BL/6,

129SvEvBrd and FVB. 9V/null mice from multiple litters were randomly assigned into

each treatment group on a rolling basis. Both male and female mice were enrolled in each

group with an attempt to balance gender in the groups. All mice were housed under

pathogen-free conditions and were monitored daily and weighed weekly. All AAV treated

mice showed normal growth and weight gain.

At the end of the study, mice were euthanized by pentobarbital (100 mg/kg). Mice were

transcardially perfused with saline. Liver, spleen and lung were then dissected.

AAV/VPRIV preparation and administration

Aliquots of AAV8-FLF-PL64 were stored at -80°C. Before injection, the aliquot was

thawed on ice and diluted with X-VIVO 10 (Lonza, pH7.4, 4°C), and gently mixed by

vortexing briefly at low speed. The diluted AAV was kept on ice before injection and used

within 2 hours.

VPRIV R was resuspended and aliquoted (25, 50, 100 (11) and stored µ1) and stored at at -80°C. -80°C. Before Before

injection, the aliquot was thawed on ice and diluted with acidified X-VIVO 10 (Lonza,

pH5.5, 4°C) to indicated dose, and gently mixed by vortexing briefly at low speed. The

diluted enzyme was kept on ice before injection and used within 2 hours.

WO wo 2020/161483 PCT/GB2020/050251 PCT/GB2020/050251

AAV (2 1012 vg/kg) X 10¹² and vg/kg) vehicle and (X-vivo) vehicle were (X-vivo) given were one given time one to to time 9V/null mice 9V/null at at mice 8 8

weeks of age with indicated doses at 5 uL/g µL/g body weight (BW). WT mice were

administrated with vehicle. AAV and vehicle administration were via tail vein to the mice

while briefly under isoflurane. VPRIV R was administered by tail vein bolus injection to

9V/null mice anesthetized with mixture of isoflurane and oxygen in bio-bubble room at

60U/kg and 2.5 uL/g µL/g BW, starting at 8 weeks of age, biweekly, for 7 injections.

Tissue collection

Blood (~100 uL) µL) was collected from tail vein in a tube containing 0.5 M EDTA (5 uL) µL) at

12 weeks, 16 weeks and 20 weeks of age. Freshly collected blood samples were kept on

ice and separated to plasma to assay for GCase activity within 2 hours. Each plasma

collection and activity assay from the VPRIV R treatment group was performed within 2

hours after the scheduled enzyme injection. A separate portion of blood (~400 uL) µL) was

processed to isolate white blood cells (WBC) for GCase activity assay. Collected WBC

was stored at -80°C.

Tissues (liver, lung, spleen, bone marrow) were collected at experimental endpoint (20

weeks of age). Tissue collection from the VPRIV R group was performed within 2 hours

after the final scheduled enzyme injection. Liver, lung and spleen samples were divided

into 4 parts, with 3 parts frozen in individual tubes and stored at -80°C prior to GCase

activity assay, protein and substrate analysis. The remaining part was fixed in 10%

Formalin for histology analysis. Bone marrow cells were collected from femurs and tibias

of both legs of the mice and frozen in two tubes stored at -80°C freezer for GCase activity

and substrate assays.

GCase activity assay

Tissues were homogenized in 1% Na taurocholate and 1% Triton X-100 (Tc/Tx) using a

Precellys Evolution tissue homogenizer for two cycles (20 seconds each, 30 seconds

interval) at 4°C. Cells (bone marrow (BM) and white blood cells (WBC)) were

homogenized in 1% Tc/Tx with sonication at 4°C. Tissue and cell lysates (2 uL) µL) were

WO wo 2020/161483 PCT/GB2020/050251 PCT/GB2020/050251

diluted (5 x) with reaction buffer in assay mixture (0.025 M Citrate-phosphate buffer,

pH5.6). Diluted lysates (10 uL) µL) (in triplicate per sample) were loaded to reaction plate.

GCase activity was determined fluorometrically with 4-methylumberlliferyl-B-D 4-methylumberlliferyl-}-D-

glucopyranoside (4MU-Glucose, 4 mM) (Biosynth AG, Switzerland) in the presence and

absence of 2 mM Conduritol B epoxide (Millipore. CA) incubated for 1 hour at 37°C.

Protein concentrations were determined using BCA Protein Assay Reagent (Pierce,

Rockford, IL).

Plasma was diluted in 0.025 M Citrate-phosphate buffer, pH5.6. GCase activity was

determined fluorometrically with 4-methylumberlliferyl-B-D-glucopyranoside (4MU-

Glucose, 4 mM) (Biosynth AG, Switzerland) as above.

Substrate analysis

Frozen tissues were weighed and homogenized in 3.6 mL of Methanol/Chloroform/H2O Methanol/Chloroform/HO

(2:1:0.6 v/v/v). Aliquots (500 uL) µL) of lysate were subjected to LC/MS analysis. The

quantitated hexosylceramide and hexosylsphingosine were normalized by tissue weight.

Plasma was diluted in water (40 uL µL plasma + 60 uL µL water) and subjected to LC/MS

analysis. Substrate level was normalized by plasma volume.

Bone marrow cells were suspended in 200 uL µL water and sonicated and vortexed to make

cell lysate. 160 uL µL lysate was subjected to LC/MS analysis. Remaining lysate was

determined for protein concentration. Substrate level was normalized by mg protein.

LC/MS analysis was performed to analyse hexosylceramide and hexosylsphingosine

concentrations. Since galatosylceramide and galatosylsphingosine levels are very low in

this mouse model model, measured hexosylceramide and hexosylsphingosine

concentrations represent levels of glucosylceramide and glucosylsphingosine, respectively.

Histology analysis

WO wo 2020/161483 PCT/GB2020/050251 PCT/GB2020/050251

Liver, lung, spleen and bone marrow were dissected from saline perfused mice and fixed in

Formalin (10%) and paraffin embedded. Fixed tissues were cut into 4 um µm sections and

mounted on slides.

Storage cell count

Tissue sections were stained with hematoxylin and eosin (H&E) by Autostainner (Leica

Autostainner XL). The stained tissues were scanned with Aperio AT2 (Leica, 40X). The

tissue images were processed with Aperio ImageScope (V12.4.0.0543). 10 photos of 20X

magnitude (500 um µm X 800 um µm image) from liver and lung per mouse were chosen for

analysis. Storage cells were counted from each image. Average of cell counts from 10

images was calculated for data graph. Definition of "storage cells" is based on the size of

cells (macrophage), e.g. size of storage cells in liver is > 10 um, µm, in lung is > 15 um. µm.

CD68 staining and quantification

Tissue sections were stained with rabbit anti-mouse CD68 antibody (1:25. Abcam

Ab53444) in Discover Ultra automated IHC/ISH slide staining machine. The tissues were

counter-stained with hematoxylin on cell nuclei. Stained tissues were scanned with Aperio

AT2 (Leica, 40X) and the images were acquired by Aperio ImageScope (V12.4.0.0543).

The images of liver and lung at 20X magnitude (500 um µm X 800 um) µm) were used for

quantitative analysis. IHC signals from 5 images of liver or lung per mouse were analysed

using Image J (Fiji, v5.1). Average CD68 signals per mouse was calculated for data graph.

Statistical analyses

The data was analysed by Student's t-test or One Way ANOVA. OneWay ANOVA. Figure Figure graphs graphs and and

statistical analysis were generated by PRISM 8 software (PRISM version 8.0.1).

2. Results

GCase activity

AAV-FLF-PL64 treatment to restore active GCase levels in 9V/null mice was studied by

measuring GCase activity in cells and tissues. White blood cells (WBC), bone marrow and

WO wo 2020/161483 PCT/GB2020/050251 PCT/GB2020/050251

tissue samples were collected at the experimental end point as above (i.e. 12 weeks post

AAV-FLF-PL64 injection or on the final VPRIV R administration), when the mice are at

20 weeks of age.

VPRIV R was shown to increase the activity across all cells and tissues tested (Figure 13).

As stated above, tissues of the VPRIV R treated group were collected within 2 hours post

last injection, and this is in line with previous data showing that this is within the period

where VPRIV R is at its C-max in the tissues.

AAV-FLF-PL64 was shown to also significantly increase GCase activity in all tissues

following only a single administration (Figure 13). Compared to Vehicle-9V/null, liver

GCase activity increased by 4.7-fold, and spleen GCase activity increased by 2.5-fold. In

white blood cells GCase activity was seen to significantly increase in the AAV-FLF-PL64

treated groups by 7-9 fold. In particular, the GCase activity level in white blood cells

reached to about 82% of WT activity levels.

Tissue Histology

Visceral pathology in 9V/null mice was determined by counting foamy macrophages as

storage cells and quantitating CD68 staining signals on activated macrophages. The storage

cells were counted in H&E stained liver sections. CD68 signal (brown colour) intensity

was quantified on anti-CD68 antibody stained liver and lung sections.

Storages cells at size 10 10um µmin inliver liverwere werecounted countedfrom from10 10images imagesper pertissues tissuesof ofeach each

mouse. In liver, the number of storage cells was undetectable in the AAV-FLF-PL64

treated groups, as well as in the VPRIV R group. (Figure 14)

CD68 signals in liver were also significantly decreased in the AAV-FLF-PL64 treated

groups. AAV-FLF-PL64 treatment reduced CD68 signals to about 25% of Vehicle-9V/null

level. In comparison, CD68 signals in VPRIV group was about 37% of Vehicle-9V/null

level. (Figure 14)

WO wo 2020/161483 PCT/GB2020/050251 PCT/GB2020/050251

Substrate accumulation

9V/null mice are known to develop glycolipid substrates accumulation in liver, lung and

spleen (Xu et al. PLoS One. 2010 May 20;5(5):e10750). For example, the study showed

that hexosylceramide in the control Vehicle-9V/null group is above WT level by 7.97-fold

in liver and 3.57-fold in spleen (data not shown).

AAV-FLF-PL64 treated groups showed significant reduction of hexosylceramide and

hexosylsphingosine in the liver and spleen compared to Vehicle-9V/null (Figure 15). In

particular, AAV-FLF-PL64 treated groups had hexosylceramide levels reduced to 1.20-

fold times the wild-type level in liver and 1.03-fold times the wild-type level in spleen

(data not shown). Similar reduction to close to the WT level was seen upon analysis of

bone marrow (data not shown).

On the other hand, VPRIV R treatment only showed a significant reduction of

hexosylceramide in liver, with the other tested tissues showing no significant changes in

the hexosylceramide levels. VPRIV R did not appear to have any significant effect on

hexosylphingosine levels in any tested tissue.

It will of course be understood that, although the present invention has been described by

way of example, the examples are in no way meant to be limiting, and modifications can

be made within the scope of the claims hereinafter. Preferred features of each embodiment

of the invention are as for each of the other embodiments mutatis mutandis. All

publications, including but not limited to patents and patent applications, cited in this

specification are herein incorporated by reference as if each individual publication was

specifically and individually indicated to be incorporated by reference herein.

SEQUENCE LISTING SEQUENCE LISTING

<110> FREELINE THERAPEUTICS LIMITED <110> FREELINE THERAPEUTICS LIMITED <120> POLYNUCLEOTIDES <120> POLYNUCLEOTIDES

<130> N414281WO <130> N414281WO

<160> 25 <160> 25

<170> PatentIn version 3.5 <170> PatentIn version 3.5

<210> 1 <210> 1 <211> 1494 <211> 1494 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic construct <223> Synthetic construct

<400> 1 <400> 1 gccaggccct gcatccctaa gagctttggc tacagctctg tggtgtgtgt gtgcaatgcc 60 gccaggccct gcatccctaa gagctttggo tacagctctg tggtgtgtgt gtgcaatgcc 60

acctactgtg acagctttga cccccccacc tttcctgccc tgggcacctt cagcagatat 120 acctactgtg acagctttga cccccccacc tttcctgccc tgggcacctt cagcagatat 120

gagagcacca ggtctgggag gaggatggag ctgagcatgg ggcccatcca ggctaatcac 180 gagagcacca ggtctgggag gaggatggag ctgagcatgg ggcccatcca ggctaatcac 180

actggcactg gcctgctgct gaccctgcag cctgagcaga agttccagaa agtaaagggc 240 actggcactg gcctgctgct gaccctgcag cctgagcaga agttccagaa agtaaagggc 240

tttggagggg ccatgactga tgctgctgct ctgaacatcc tggccctgag cccccctgcc 300 tttggagggg ccatgactga tgctgctgct ctgaacatcc tggccctgag cccccctgcc 300

cagaatctgc tgctgaagag ctacttctct gaggagggca ttggctataa catcatcagg 360 cagaatctgc tgctgaagag ctacttctct gaggagggca ttggctataa catcatcagg 360

gtgcccatgg ccagctgtga cttcagcatc aggacctaca cctatgctga cacccctgat 420 gtgcccatgg ccagctgtga cttcagcatc aggacctaca cctatgctga cacccctgat 420

gatttccagc tgcacaactt cagcctgcct gaggaggata ccaagctgaa gatcccactg 480 gatttccagc tgcacaactt cagcctgcct gaggaggata ccaagctgaa gatcccactg 480

atccacaggg ctctgcagct ggcccagagg cctgtgagcc tgctggccag cccctggacc 540 atccacaggg ctctgcagct ggcccagagg cctgtgagcc tgctggccag cccctggacc 540

agccccactt ggctgaagac caatggggct gtgaatggga aggggagcct gaagggacag 600 agccccactt ggctgaagac caatggggct gtgaatggga aggggagcct gaagggacag 600

cctggagaca tctaccacca gacctgggcc agatactttg tgaagttcct ggatgcctat 660 cctggagaca tctaccacca gacctgggcc agatactttg tgaagttcct ggatgcctat 660

gctgagcaca agctgcagtt ctgggctgtg actgctgaga atgagccttc tgctgggctg 720 gctgagcaca agctgcagtt ctgggctgtg actgctgaga atgagccttc tgctgggctg 720

ctgtctggct accccttcca atgcctgggc ttcacccctg agcatcagag ggacttcatt 780 ctgtctggct accccttcca atgcctgggc ttcacccctg agcatcagag ggacttcatt 780

gccagggacc tgggccctac cctggccaac agcactcacc ataatgttag gctgctgatg 840 gccagggacc tgggccctac cctggccaac agcactcacc ataatgttag gctgctgatg 840

ctggatgacc agaggctgct gctgccccac tgggctaagg tggtgctgac tgaccctgag 900 ctggatgacc agaggctgct gctgccccac tgggctaagg tggtgctgac tgaccctgag 900

gctgctaaat atgtgcatgg cattgctgtg cattggtacc tggactttct ggctcctgcc 960 gctgctaaat atgtgcatgg cattgctgtg cattggtacc tggactttct ggctcctgcc 960

aaggccaccc tgggggagac ccacaggctg ttccccaaca ccatgctgtt tgcctctgag 1020 aaggccaccc tgggggagac ccacaggctg ttccccaaca ccatgctgtt tgcctctgag 1020 gcctgtgtgg gcagcaagtt ctgggagcag tctgtgaggc tgggcagctg ggataggggg 1080 gcctgtgtgg gcagcaagtt ctgggagcag tctgtgaggc tgggcagctg ggataggggg 1080 atgcagtaca gccacagcat catcaccaac ctgctgtacc atgtggtggg ctggactgac 1140 atgcagtaca gccacagcat catcaccaac ctgctgtacc atgtggtggg ctggactgac 1140 tggaacctgg ccctgaaccc tgagggagga cctaactggg tcagaaactt tgtggacagc 1200 tggaacctgg ccctgaaccc tgagggagga cctaactggg tcagaaactt tgtggacagc 1200 cccatcattg tggacatcac caaggacacc ttttacaagc agcccatgtt ctaccacctg 1260 cccatcattg tggacatcac caaggacacc ttttacaagc agcccatgtt ctaccacctg 1260 ggccacttca gcaagttcat ccctgagggc agccagagag tggggctggt ggccagccag 1320 ggccacttca gcaagttcat ccctgagggc agccagagag tggggctggt ggccagccag 1320 aagaatgacc tggatgctgt ggctctgatg catcctgatg gctctgctgt ggtggtggtg 1380 aagaatgacc tggatgctgt ggctctgatg catcctgatg gctctgctgt ggtggtggtg 1380 ctgaacagga gctctaagga tgtgcctctg accatcaagg atcctgctgt gggcttcctg 1440 ctgaacagga gctctaagga tgtgcctctg accatcaagg atcctgctgt gggcttcctg 1440 gagaccatca gccctggcta cagcatccac acctacctgt ggaggaggca gtga 1494 gagaccatca gccctggcta cagcatccac acctacctgt ggaggaggca gtga 1494

<210> 2 <210> 2 <211> 1494 <211> 1494 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic construct <223> Synthetic construct

<400> 2 <400> 2 gccaggccct gtatccctaa gagctttggc tacagctcag tagtttgtgt ctgtaatgcc 60 gccaggccct gtatccctaa gagctttggc tacagctcag tagtttgtgt ctgtaatgcc 60

acatactgtg actcctttga cccccctacc ttccctgccc tgggaacctt cagcagatat 120 acatactgtg actcctttga cccccctacc ttccctgccc tgggaacctt cagcagatat 120

gagtcaacaa gatcaggaag gaggatggag ctgtcaatgg gacccatcca ggctaatcac 180 gagtcaacaa gatcaggaag gaggatggag ctgtcaatgg gacccatcca ggctaatcac 180

acaggcacag gcctgctgct gaccctgcag ccagaacaga agttccagaa agtgaaggga 240 acaggcacag gcctgctgct gaccctgcag ccagaacaga agttccagaa agtgaaggga 240

tttggaggag ccatgacaga tgctgctgct ctcaacatcc tggccctgtc accccctgcc 300 tttggaggag ccatgacaga tgctgctgct ctcaacatcc tggccctgtc accccctgcc 300

cagaatctgc tgctgaagtc atacttctct gaagaaggaa ttggatataa catcatcagg 360 cagaatctgc tgctgaagtc atacttctct gaagaaggaa ttggatataa catcatcagg 360

gtgcccatgg ccagctgtga cttctccatc aggacctaca cctatgctga cacccctgat 420 gtgcccatgg ccagctgtga cttctccatc aggacctaca cctatgctga cacccctgat 420

gatttccagc tgcacaactt cagcctccca gaggaagata ccaagctcaa gatccctctg 480 gatttccagc tgcacaactt cagcctccca gaggaagata ccaagctcaa gatccctctg 480

atacataggg cactgcagct ggcccagagg cctgtgtcac tcctggccag cccctggaca 540 atacataggg cactgcagct ggcccagagg cctgtgtcac tcctggccag cccctggaca 540

tcacccactt ggctcaagac caatggagct gtgaatggaa agggatcact caagggacag 600 tcacccactt ggctcaagac caatggagct gtgaatggaa agggatcact caagggacag 600

gctgagcaca agctgcagtt ctgggcagtg acagctgaaa atgagccttc tgctggactg 720 gctgagcaca agctgcagtt ctgggcagtg acagctgaaa atgagccttc tgctggactg 720

ctgtcaggat accccttcca gtgtctgggc ttcacccctg aacatcagag ggacttcatt 780 ctgtcaggat accccttcca gtgtctgggc ttcacccctg aacatcagag ggacttcatt 780

gccagggacc tgggacctac ccttgccaac tcaactcacc acaatgtcag gctgctcatg 840 gccagggacc tgggacctac ccttgccaac tcaactcacc acaatgtcag gctgctcatg 840 ctggatgacc agaggctgct gctgccccac tgggccaagg tggtgctgac agacccagaa 900 006 gctgctaaat atgtgcatgg cattgctgtg cattggtacc tggacttcct ggctccagcc 960 096 aaggccaccc tgggagagac acacaggctg ttccccaaca ccatgctctt tgcctctgag 1020 0201 gcctgtgtgg gctccaagtt ctgggagcag tcagtgaggc tgggctcctg ggatagggga 1080 080I atgcagtaca gccacagcat catcacaaac ctcctgtacc atgtggtggg ctggactgac 1140 e tggaacctgg ccctgaaccc tgaaggagga cccaactggg tcagaaattt tgtggactca 1200 cccatcattg tggacatcac caaggacaca ttctacaagc agcccatgtt ctaccacctg 1260 e ggccacttca gcaagttcat ccctgagggc tcccagaggg tgggactggt ggcctcacag 1320 OZET aagaatgacc tggatgcagt ggccctgatg catcctgatg gctctgctgt ggtggttgtg 1380 8787788188 08ET ctgaatagat cctctaagga tgtgcctctg accatcaagg atcctgctgt gggcttcctg 1440 DATE gagacaatct cacctggcta ctccatccac acctacctgt ggaggaggca gtga 1494

<210> 3 <0IZ> E <211> 1494 <IIZ> <212> DNA <ZIZ> <EIZ>

<220> <022> ANC <213> Artificial Sequence and <223> Synthetic construct <EZZ>

<400> 3 E <00 e gccaggccct gcatccctaa gagctttggc tacagctctg tggtgtgtgt gtgcaatgcc 60 09

acatactgtg actcctttga cccccccacc tttcctgccc tgggcacatt ctccagatat 120 OZI

gagagcacaa gatctgggag aaggatggag ctgagcatgg ggcccatcca ggctaatcac 180 08T

actggcacag gcctgctgct gaccctgcag cctgaacaga agtttcagaa agtgaaggga 240

tttggagggg ccatgacaga tgctgctgct ctgaatatcc tggccctgtc accccctgcc 300 00E

the e cagaatctgc tgctgaagag ctacttttca gaagaaggaa ttggatataa tatcatcaga 360

7 09E

gtgcccatgg ccagctgtga cttttccatc agaacctaca cctatgcaga cacccctgat 420

gattttcagc tgcacaattt tagcctgcct gaggaagata ccaagctgaa gatacccctg 480 08/

attcacaggg ccctgcagct ggcccagagg cctgtttcac tgctggccag cccctggaca 540

the tcacccacct ggctgaagac caatggagct gtgaatggga aggggtcact gaagggacag 600 009

the cctggagaca tctaccacca gacctgggcc agatactttg tgaagtttct ggatgcctat 660 gctgagcaca agctgcagtt ttgggcagtg acagctgaaa atgagccttc agctgggctg 720 ctgtcaggat acccctttca gtgcctgggc tttacccctg aacatcagag ggactttatt 780 gccagggacc tgggccctac cctggccaat agcacccacc ataatgtgag gttgctgatg 840 ctggatgacc agaggctgct gctgccccac tgggcaaagg tggtgctgac agaccctgaa 900 gcagctaaat atgttcatgg cattgctgtg cattggtacc tggactttct ggctcctgcc 960 aaggccaccc tgggggagac acacaggctg tttcccaata ccatgctgtt tgcctctgag 1020 gcctgtgtgg gctccaagtt ttgggagcag tctgtgaggc tgggctcctg ggatagaggg 1080 atgcagtaca gccacagcat catcaccaat ctgctgtacc atgtggtggg ctggactgac 1140 tggaatctgg ccctgaatcc tgaaggagga cctaactggg tcaggaattt tgtggacagc 1200 cccatcattg tggacatcac caaggacacc ttttacaagc agcccatgtt ttaccacctg 1260 ggccacttta gcaagtttat tcctgagggc tcccagagag tggggctggt tgccagccag 1320 aagaatgacc tggatgcagt ggcactgatg catcctgatg gctcagctgt tgtggtggtg 1380 ctgaatagat ccagcaagga tgtgcctctg accatcaagg atcctgctgt gggctttctg 1440 gagacaatct cacctggcta ctccattcac acctacctgt ggagaaggca gtga 1494

<210> 4 <211> 1494 <212> DNA <213> Artificial Sequence

<220> <223> Synthetic construct

<400> 4 gccaggcctt gcatcccaaa gtctttcggc tacagctccg tggtgtgcgt gtgcaacgcc 60

acctattgtg actccttcga tccccctacc tttcccgccc tgggcacatt ttctagatac 120

gagtctacac gcagcggccg gagaatggag ctgagcatgg gccctatcca ggccaatcac 180

acaggaacag gcctgctgct gaccctgcag ccagagcaga agttccagaa ggtgaagggc 240

tttggcggag ccatgacaga tgcagccgcc ctgaacatcc tggccctgtc cccacccgcc 300

cagaatctgc tgctgaagtc ctacttctct gaggagggca tcggctataa catcatccgg 360

gtgcccatgg ccagctgcga cttttccatc agaacctaca catatgccga tacccctgac 420

gatttccagc tgcacaattt ttccctgcca gaggaggata caaagctgaa gatccccctg 480 attcaccggg ccctgcagct ggcacagcgg cccgtgagcc tgctggccag cccctggacc 540 attcaccggg ccctgcagct ggcacagcgg cccgtgagcc tgctggccag cccctggacc 540 tcccctacat ggctgaagac caacggcgcc gtgaatggca agggctctct gaagggacag 600 tcccctacat ggctgaagac caacggcgcc gtgaatggca agggctctct gaagggacag 600 cctggcgaca tctaccacca gacatgggcc agatatttcg tgaagtttct ggatgcctac 660 cctggcgaca tctaccacca gacatgggcc agatatttcg tgaagtttct ggatgcctac 660 gccgagcaca agctgcagtt ctgggccgtg acagcagaga atgagccttc tgccggcctg 720 gccgagcaca agctgcagtt ctgggccgtg acagcagaga atgagccttc tgccggcctg 720 ctgagcggct atcccttcca gtgcctgggc tttacacctg agcaccagcg ggactttatc 780 ctgagcggct atcccttcca gtgcctgggc tttacacctg agcaccagcg ggactttatc 780 gccagagatc tgggcccaac cctggccaac tccacacacc acaatgtgag gctgctgatg 840 gccagagatc tgggcccaac cctggccaac tccacacacc acaatgtgag gctgctgatg 840 ctggacgatc agcgcctgct gctgcctcac tgggccaagg tggtgctgac cgacccagag 900 ctggacgatc agcgcctgct gctgcctcac tgggccaagg tggtgctgac cgacccagag 900 gccgccaagt acgtgcacgg catcgccgtg cactggtatc tggatttcct ggcacctgca 960 gccgccaagt acgtgcacgg catcgccgtg cactggtatc tggatttcct ggcacctgca 960 aaggccaccc tgggagagac acaccggctg ttccctaaca ccatgctgtt tgccagcgag 1020 aaggccaccc tgggagagac acaccggctg ttccctaaca ccatgctgtt tgccagcgag 1020 gcctgcgtgg gctccaagtt ttgggagcag tccgtgaggc tgggatcttg ggacagaggc 1080 gcctgcgtgg gctccaagtt ttgggagcag tccgtgaggc tgggatcttg ggacagaggc 1080 atgcagtact cccactctat catcaccaat ctgctgtatc acgtggtggg ctggacagac 1140 atgcagtact cccactctat catcaccaat ctgctgtatc acgtggtggg ctggacagac 1140 tggaacctgg ccctgaatcc agagggcggc cccaactggg tgagaaattt cgtggatagc 1200 tggaacctgg ccctgaatcc agagggcggc cccaactggg tgagaaattt cgtggatagc 1200 cccatcatcg tggacatcac caaggataca ttctacaagc agccaatgtt ttatcacctg 1260 cccatcatcg tggacatcac caaggataca ttctacaagc agccaatgtt ttatcacctg 1260 ggccacttct ctaagtttat ccctgagggc agccagaggg tgggcctggt ggccagccag 1320 ggccacttct ctaagtttat ccctgagggc agccagaggg tgggcctggt ggccagccag 1320 aagaacgacc tggatgccgt ggccctgatg caccctgatg gctccgccgt ggtggtggtg 1380 aagaacgacc tggatgccgt ggccctgatg caccctgatg gctccgccgt ggtggtggtg 1380 ctgaatcgct ctagcaagga cgtgcctctg accatcaagg atccagccgt gggatttctg 1440 ctgaatcgct ctagcaagga cgtgcctctg accatcaagg atccagccgt gggatttctg 1440 gagactattt cacctggcta ttcaattcat acctacctgt ggaggaggca gtga 1494 gagactattt cacctggcta ttcaattcat acctacctgt ggaggaggca gtga 1494

<210> 5 <210> 5 <211> 1611 <211> 1611 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic construct <223> Synthetic construct

<400> 5 <400> 5 atggagtttt caagtccttc cagagaggaa tgtcccaagc ctttgagtag ggtaagcatc 60 atggagtttt caagtccttc cagagaggaa tgtcccaagc ctttgagtag ggtaagcatc 60

atggctggca gcctcacagg attgcttcta cttcaggcag tgtcgtgggc atcaggtgcc 120 atggctggca gcctcacagg attgcttcta cttcaggcag tgtcgtgggc atcaggtgcc 120

aggccctgca tccctaagag ctttggctac agctctgtgg tgtgtgtgtg caatgccacc 180 aggccctgca tccctaagag ctttggctac agctctgtgg tgtgtgtgtg caatgccacc 180

tactgtgaca gctttgaccc ccccaccttt cctgccctgg gcaccttcag cagatatgag 240 tactgtgaca gctttgaccc ccccaccttt cctgccctgg gcaccttcag cagatatgag 240

agcaccaggt ctgggaggag gatggagctg agcatggggc ccatccaggc taatcacact 300 agcaccaggt ctgggaggag gatggagctg agcatggggc ccatccaggc taatcacact 300 ggcactggcc tgctgctgac cctgcagcct gagcagaagt tccagaaagt aaagggcttt 360 09E ggaggggcca tgactgatgc tgctgctctg aacatcctgg ccctgagccc ccctgcccag 420 aatctgctgc tgaagagcta cttctctgag gagggcattg gctataacat catcagggtg 480 08/ e cccatggcca gctgtgactt cagcatcagg acctacacct atgctgacac ccctgatgat 540 ttccagctgc acaacttcag cctgcctgag gaggatacca agctgaagat cccactgatc 600 009 cacagggctc tgcagctggc ccagaggcct gtgagcctgc tggccagccc ctggaccagc 660 099 cccacttggc tgaagaccaa tggggctgtg aatgggaagg ggagcctgaa gggacagcct 720 02L ggagacatct accaccagac ctgggccaga tactttgtga agttcctgga tgcctatgct 780 08L gagcacaagc tgcagttctg ggctgtgact gctgagaatg agccttctgc tgggctgctg 840 tctggctacc ccttccaatg cctgggcttc acccctgagc atcagaggga cttcattgcc 900 006 agggacctgg gccctaccct ggccaacagc actcaccata atgttaggct gctgatgctg 960 096 e gatgaccaga ggctgctgct gccccactgg gctaaggtgg tgctgactga ccctgaggct 1020 gctaaatatg tgcatggcat tgctgtgcat tggtacctgg actttctggc tcctgccaag 1080 080T gccaccctgg gggagaccca caggctgttc cccaacacca tgctgtttgc ctctgaggcc 1140 tgtgtgggca gcaagttctg ggagcagtct gtgaggctgg gcagctggga tagggggatg 1200 cagtacagcc acagcatcat caccaacctg ctgtaccatg tggtgggctg gactgactgg 1260 092T aacctggccc tgaaccctga gggaggacct aactgggtca gaaactttgt ggacagcccc 1320 OZET atcattgtgg acatcaccaa ggacaccttt tacaagcagc ccatgttcta ccacctgggc 1380 08ET cacttcagca agttcatccc tgagggcagc cagagagtgg ggctggtggc cagccagaag 1440 aatgacctgg atgctgtggc tctgatgcat cctgatggct ctgctgtggt ggtggtgctg 1500 00ST aacaggagct ctaaggatgt gcctctgacc atcaaggatc ctgctgtggg cttcctggag 1560 09ST accatcagcc ctggctacag catccacacc tacctgtgga ggaggcagtg a 1611 e TITI

<210> 6 <0IZ> 9 <211> 1611 <IIZ> <212> DNA <ZIZ> ANC <213> Artificial Sequence <ETZ>

<220> <022> <223> Synthetic construct <EZZ>

<400> 6 9 <00 atggagtttt caagtccttc cagagaggaa tgtcccaagc ctttgagtag ggtaagcatc 60 09

atggctggca gcctcacagg attgcttcta cttcaggcag tgtcgtgggc atcaggtgcc 120 OZI

aggccctgta tccctaagag ctttggctac agctcagtag tttgtgtctg taatgccaca 180 08T

tactgtgact cctttgaccc ccctaccttc cctgccctgg gaaccttcag cagatatgag 240

tcaacaagat caggaaggag gatggagctg tcaatgggac ccatccaggc taatcacaca 300 00E

ggcacaggcc tgctgctgac cctgcagcca gaacagaagt tccagaaagt gaagggattt 360 09E

ggaggagcca tgacagatgc tgctgctctc aacatcctgg ccctgtcacc ccctgcccag 420

aatctgctgc tgaagtcata cttctctgaa gaaggaattg gatataacat catcagggtg 480 08/

cccatggcca gctgtgactt ctccatcagg acctacacct atgctgacac ccctgatgat 540

ttccagctgc acaacttcag cctcccagag gaagatacca agctcaagat ccctctgata 600 009

catagggcac tgcagctggc ccagaggcct gtgtcactcc tggccagccc ctggacatca 660 099

cccacttggc tcaagaccaa tggagctgtg aatggaaagg gatcactcaa gggacagcct 720 OZL

ggagacatct accaccagac ctgggccaga tactttgtga agttcctgga tgcctatgct 780 08L

gagcacaagc tgcagttctg ggcagtgaca gctgaaaatg agccttctgc tggactgctg 840 78 tcaggatacc ccttccagtg tctgggcttc acccctgaac atcagaggga cttcattgcc 900 006

agggacctgg gacctaccct tgccaactca actcaccaca atgtcaggct gctcatgctg 960 096

gatgaccaga ggctgctgct gccccactgg gccaaggtgg tgctgacaga cccagaagct 1020 0201

gctaaatatg tgcatggcat tgctgtgcat tggtacctgg acttcctggc tccagccaag 1080 080I

gccaccctgg gagagacaca caggctgttc cccaacacca tgctctttgc ctctgaggcc 1140

tgtgtgggct ccaagttctg ggagcagtca gtgaggctgg gctcctggga taggggaatg 1200

cagtacagcc acagcatcat cacaaacctc ctgtaccatg tggtgggctg gactgactgg 1260

aacctggccc tgaaccctga aggaggaccc aactgggtca gaaattttgt ggactcaccc 1320 787777eee8 OZET

atcattgtgg acatcaccaa ggacacattc tacaagcagc ccatgttcta ccacctgggc 1380 the the 08EI

cacttcagca agttcatccc tgagggctcc cagagggtgg gactggtggc ctcacagaag 1440

aatgacctgg atgcagtggc cctgatgcat cctgatggct ctgctgtggt ggttgtgctg 1500 00ST

aatagatcct ctaaggatgt gcctctgacc atcaaggatc ctgctgtggg cttcctggag 1560 09ST

acaatctcac ctggctactc catccacacc tacctgtgga ggaggcagtg a 1611 e

<210> 7 <210> 7 <211> 1611 <211> 1611 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic construct <223> Synthetic construct

<400> 7 <400> 7 atggagtttt caagtccttc cagagaggaa tgtcccaagc ctttgagtag ggtaagcatc 60 atggagtttt caagtccttc cagagaggaa tgtcccaagc ctttgagtag ggtaagcatc 60

aggccctgca tccctaagag ctttggctac agctctgtgg tgtgtgtgtg caatgccaca 180 aggccctgca tccctaagag ctttggctac agctctgtgg tgtgtgtgtg caatgccaca 180

tactgtgact cctttgaccc ccccaccttt cctgccctgg gcacattctc cagatatgag 240 tactgtgact cctttgaccc ccccaccttt cctgccctgg gcacattctc cagatatgag 240

agcacaagat ctgggagaag gatggagctg agcatggggc ccatccaggc taatcacact 300 agcacaagat ctgggagaag gatggagctg agcatggggc ccatccaggc taatcacact 300

ggcacaggcc tgctgctgac cctgcagcct gaacagaagt ttcagaaagt gaagggattt 360 ggcacaggcc tgctgctgac cctgcagcct gaacagaagt ttcagaaagt gaagggattt 360

ggaggggcca tgacagatgc tgctgctctg aatatcctgg ccctgtcacc ccctgcccag 420 ggaggggcca tgacagatgc tgctgctctg aatatcctgg ccctgtcacc ccctgcccag 420

aatctgctgc tgaagagcta cttttcagaa gaaggaattg gatataatat catcagagtg 480 aatctgctgc tgaagagcta cttttcagaa gaaggaattg gatataatat catcagagtg 480

cccatggcca gctgtgactt ttccatcaga acctacacct atgcagacac ccctgatgat 540 cccatggcca gctgtgactt ttccatcaga acctacacct atgcagacac ccctgatgat 540

tttcagctgc acaattttag cctgcctgag gaagatacca agctgaagat acccctgatt 600 tttcagctgc acaattttag cctgcctgag gaagatacca agctgaagat acccctgatt 600

cacagggccc tgcagctggc ccagaggcct gtttcactgc tggccagccc ctggacatca 660 cacagggccc tgcagctggc ccagaggcct gtttcactgc tggccagccc ctggacatca 660

cccacctggc tgaagaccaa tggagctgtg aatgggaagg ggtcactgaa gggacagcct 720 cccacctggc tgaagaccaa tggagctgtg aatgggaagg ggtcactgaa gggacagcct 720

ggagacatct accaccagac ctgggccaga tactttgtga agtttctgga tgcctatgct 780 ggagacatct accaccagac ctgggccaga tactttgtga agtttctgga tgcctatgct 780

gagcacaagc tgcagttttg ggcagtgaca gctgaaaatg agccttcagc tgggctgctg 840 gagcacaagc tgcagttttg ggcagtgaca gctgaaaatg agccttcagc tgggctgctg 840

tcaggatacc cctttcagtg cctgggcttt acccctgaac atcagaggga ctttattgcc 900 tcaggatacc cctttcagtg cctgggcttt acccctgaac atcagaggga ctttattgcc 900

agggacctgg gccctaccct ggccaatagc acccaccata atgtgaggtt gctgatgctg 960 agggacctgg gccctaccct ggccaatagc acccaccata atgtgaggtt gctgatgctg 960

gatgaccaga ggctgctgct gccccactgg gcaaaggtgg tgctgacaga ccctgaagca 1020 gatgaccaga ggctgctgct gccccactgg gcaaaggtgg tgctgacaga ccctgaagca 1020

gctaaatatg ttcatggcat tgctgtgcat tggtacctgg actttctggc tcctgccaag 1080 gctaaatatg ttcatggcat tgctgtgcat tggtacctgg actttctggc tcctgccaag 1080

gccaccctgg gggagacaca caggctgttt cccaatacca tgctgtttgc ctctgaggcc 1140 gccaccctgg gggagacaca caggctgttt cccaatacca tgctgtttgc ctctgaggcc 1140

tgtgtgggct ccaagttttg ggagcagtct gtgaggctgg gctcctggga tagagggatg 1200 tgtgtgggct ccaagttttg ggagcagtct gtgaggctgg gctcctggga tagagggatg 1200

cagtacagcc acagcatcat caccaatctg ctgtaccatg tggtgggctg gactgactgg 1260 cagtacagcc acagcatcat caccaatctg ctgtaccatg tggtgggctg gactgactgg 1260

aatctggccc tgaatcctga aggaggacct aactgggtca ggaattttgt ggacagcccc 1320 aatctggccc tgaatcctga aggaggacct aactgggtca ggaattttgt ggacagcccc 1320 atcattgtgg ccacctgggc atcattgtgg acatcaccaa ggacaccttt tacaagcagc ccatgtttta ccacctgggc 1380 1380 cactttagca cagccagaag cactttagca agtttattcc tgagggctcc cagagagtgg ggctggttgc cagccagaag 1440 1440 aatgacctgg aatgacctgg atgcagtggc actgatgcat cctgatggct cagctgttgt ggtggtgctg 1500 1500 aatagatcca ctttctggag aatagatcca gcaaggatgt gcctctgacc atcaaggatc ctgctgtggg ctttctggag 1560 1560 acaatctcac ctggctactc cattcacacc tacctgtgga gaaggcagtg acaatctcac ctggctactc cattcacacc tacctgtgga gaaggcagtg a 1611 1611 a

<210> 8 <210> 8 <211> 1611 <211> 1611 <212> DNA <212> DNA Artificial Sequence <213> Artificial Sequence <213>

<220> <220> Synthetic construct <223> Synthetic construct <223>

<400> 8 <400> 8 atggagtttt atggagtttt caagtccttc cagagaggaa tgtcccaagc ctttgagtag ggtaagcatc 60 60 atggctggca atcaggtgcc atggctggca gcctcacagg attgcttcta cttcaggcag tgtcgtgggc atcaggtgcc 120 120 aggccttgca aggccttgca tcccaaagtc tttcggctac agctccgtgg tgtgcgtgtg caacgccacc 180 180 tattgtgact ccctaccttt tattgtgact ccttcgatcc ccctaccttt cccgccctgg gcacattttc tagatacgag 240 240

tctacacgca agcatgggcc tctacacgca gcggccggag aatggagctg agcatgggcc ctatccaggc caatcacaca 300 300

ggaacaggcc ggaacaggcc tgctgctgac cctgcagcca gagcagaagt tccagaaggt gaagggcttt 360 360 ggcggagcca agccgccctg ggcggagcca tgacagatgc agccgccctg aacatcctgg ccctgtcccc acccgcccag 420 420

aatctgctgc tgaagtccta catccgggtg aatctgctgc tgaagtccta cttctctgag gagggcatcg gctataacat catccgggtg 480 480

cccatggcca gctgcgactt ccctgacgat cccatggcca gctgcgactt ttccatcaga acctacacat atgccgatac ccctgacgat 540 540

ttccagctgc ccccctgatt ttccagctgc acaatttttc cctgccagag gaggatacaa agctgaagat ccccctgatt 600 600

caccgggccc tgcagctggc caccgggccc tgcagctggc acagcggccc gtgagcctgc tggccagccc ctggacctcc 660 660

cctacatggc cggcgccgtg cctacatggc tgaagaccaa cggcgccgtg aatggcaagg gctctctgaa gggacagcct 720 720 ggcgacatct tatttcgtga tgcctacgcc ggcgacatct accaccagac atgggccaga tatttcgtga agtttctgga tgcctacgcc 780 780 gagcacaagc agccttctgc gagcacaagc tgcagttctg ggccgtgaca gcagagaatg agccttctgc cggcctgctg 840 840 agcggctatc agcggctatc ccttccagtg cctgggcttt acacctgagc accagcggga ctttatcgcc 900 900 agagatctgg gcccaaccct acacaccaca atgtgaggct agagatctgg gcccaaccct ggccaactcc acacaccaca atgtgaggct gctgatgctg 960 960

gacgatcagc gcctgctgct gcctcactgg gccaaggtgg tgctgaccga cccagaggcc gacgatcagc gcctgctgct gcctcactgg gccaaggtgg tgctgaccga cccagaggcc 1020 gccaagtacg tgcacggcat cgccgtgcac tggtatctgg atttcctggc acctgcaaag 1080 gccaaattacg tgcacggcat cgccgtgcac tggtatctgg atttcctggc acctgcaaag 1080 gccaccctgg gagagacaca ccggctgttc cctaacacca tgctgtttgc cagcgaggcc 1140 gccaccctgg gagagacaca ccggctgttc cctaacacca tgctgtttgc cagcgaggcc 1140 tgcgtgggct ccaagttttg ggagcagtcc gtgaggctgg gatcttggga cagaggcatg 1200 tgcgtgggct ccaagttttg ggagcagtcc gtgaggctgg gatcttggga cagaggcatg 1200 cagtactccc actctatcat caccaatctg ctgtatcacg tggtgggctg gacagactgg 1260 cagtactccc actctatcat caccaatctg ctgtatcacg tggtgggctg gacagactgg 1260 aacctggccc tgaatccaga gggcggcccc aactgggtga gaaatttcgt ggatagcccc 1320 aacctggccc tgaatccaga gggcggcccc aactgggtga gaaatttcgt ggatagcccc 1320 atcatcgtgg acatcaccaa ggatacattc tacaagcagc caatgtttta tcacctgggc 1380 atcatcgtgg acatcaccaa ggatacatto tacaagcagc caatgtttta tcacctgggc 1380 cacttctcta agtttatccc tgagggcagc cagagggtgg gcctggtggc cagccagaag 1440 cacttctcta agtttatccc tgagggcagc cagagggtgg gcctggtggc cagccagaag 1440 aacgacctgg atgccgtggc cctgatgcac cctgatggct ccgccgtggt ggtggtgctg 1500 aacgacctgg atgccgtggc cctgatgcac cctgatggct ccgccgtggt ggtggtgctg 1500 aatcgctcta gcaaggacgt gcctctgacc atcaaggatc cagccgtggg atttctggag 1560 aatcgctcta gcaaggacgt gcctctgacc atcaaggatc cagccgtggg atttctggag 1560 actatttcac ctggctattc aattcatacc tacctgtgga ggaggcagtg a 1611 actatttcac ctggctattc aattcatacc tacctgtgga ggaggcagtg a 1611

<210> 9 <210> 9 <211> 1611 <211> 1611 <212> DNA <212> DNA <213> Homo sapiens <213> Homo sapiens

<400> 9 <400> 9 atggagtttt caagtccttc cagagaggaa tgtcccaagc ctttgagtag ggtaagcatc 60 atggagtttt caagtccttc cagagaggaa tgtcccaagc ctttgagtag ggtaagcatc 60

cgcccctgca tccctaaaag cttcggctac agctcggtgg tgtgtgtctg caatgccaca 180 cgcccctgca tccctaaaag cttcggctac agctcggtgg tgtgtgtctg caatgccaca 180

tactgtgact cctttgaccc cccgaccttt cctgcccttg gtaccttcag ccgctatgag 240 tactgtgact cctttgacco cccgaccttt cctgcccttg gtaccttcag ccgctatgag 240

agtacacgca gtgggcgacg gatggagctg agtatggggc ccatccaggc taatcacacg 300 agtacacgca gtgggcgacg gatggagctg agtatggggc ccatccaggc taatcacacg 300

ggcacaggcc tgctactgac cctgcagcca gaacagaagt tccagaaagt gaagggattt 360 ggcacaggcc tgctactgac cctgcagcca gaacagaagt tccagaaagt gaagggattt 360

ggaggggcca tgacagatgc tgctgctctc aacatccttg ccctgtcacc ccctgcccaa 420 ggaggggcca tgacagatgc tgctgctctc aacatccttg ccctgtcacc ccctgcccaa 420

aatttgctac ttaaatcgta cttctctgaa gaaggaatcg gatataacat catccgggta 480 aatttgctac ttaaatcgta cttctctgaa gaaggaatcg gatataacat catccgggta 480

cccatggcca gctgtgactt ctccatccgc acctacacct atgcagacac ccctgatgat 540 cccatggcca gctgtgactt ctccatccgc acctacacct atgcagacac ccctgatgat 540

ttccagttgc acaacttcag cctcccagag gaagatacca agctcaagat acccctgatt 600 ttccagttgc acaacttcag cctcccagag gaagatacca agctcaagat acccctgatt 600

caccgagccc tgcagttggc ccagcgtccc gtttcactcc ttgccagccc ctggacatca 660 caccgagccc tgcagttggc ccagcgtccc gtttcactcc ttgccagccc ctggacatca 660

cccacttggc tcaagaccaa tggagcggtg aatgggaagg ggtcactcaa gggacagccc 720 cccacttggc tcaagaccaa tggagcggtg aatgggaagg ggtcactcaa gggacagccc 720

ggagacatct accaccagac ctgggccaga tactttgtga agttcctgga tgcctatgct 780 ggagacatct accaccagad ctgggccaga tactttgtga agttcctgga tgcctatgct 780 gagcacaagt tacagttctg ggcagtgaca gctgaaaatg agccttctgc tgggctgttg 840 gagcacaagt tacagttctg ggcagtgaca gctgaaaatg agccttctgc tgggctgttg 840 agtggatacc ccttccagtg cctgggcttc acccctgaac atcagcgaga cttcattgcc 900 agtggatacc ccttccagtg cctgggcttc acccctgaac atcagcgaga cttcattgcc 900 cgtgacctag gtcctaccct cgccaacagt actcaccaca atgtccgcct actcatgctg 960 cgtgacctag gtcctaccct cgccaacagt actcaccaca atgtccgcct actcatgctg 960 gatgaccaac gcttgctgct gccccactgg gcaaaggtgg tactgacaga cccagaagca 1020 gatgaccaac gcttgctgct gccccactgg gcaaaggtgg tactgacaga cccagaagca 1020 gctaaatatg ttcatggcat tgctgtacat tggtacctgg actttctggc tccagccaaa 1080 gctaaatatg ttcatggcat tgctgtacat tggtacctgg actttctggc tccagccaaa 1080 gccaccctag gggagacaca ccgcctgttc cccaacacca tgctctttgc ctcagaggcc 1140 gccaccctag gggagacaca ccgcctgttc cccaacacca tgctctttgc ctcagaggcc 1140 tgtgtgggct ccaagttctg ggagcagagt gtgcggctag gctcctggga tcgagggatg 1200 tgtgtgggct ccaagttctg ggagcagagt gtgcggctag gctcctggga tcgagggatg 1200 cagtacagcc acagcatcat cacgaacctc ctgtaccatg tggtcggctg gaccgactgg 1260 cagtacagcc acagcatcat cacgaacctc ctgtaccatg tggtcggctg gaccgactgg 1260 aaccttgccc tgaaccccga aggaggaccc aattgggtgc gtaactttgt cgacagtccc 1320 aaccttgccc tgaaccccga aggaggaccc aattgggtgc gtaactttgt cgacagtccc 1320 atcattgtag acatcaccaa ggacacgttt tacaaacagc ccatgttcta ccaccttggc 1380 atcattgtag acatcaccaa ggacacgttt tacaaacagc ccatgttcta ccaccttggc 1380 cacttcagca agttcattcc tgagggctcc cagagagtgg ggctggttgc cagtcagaag 1440 cacttcagca agttcattcc tgagggctcc cagagagtgg ggctggttgo cagtcagaag 1440 aacgacctgg acgcagtggc actgatgcat cccgatggct ctgctgttgt ggtcgtgcta 1500 aacgacctgg acgcagtggc actgatgcat cccgatggct ctgctgttgt ggtcgtgcta 1500 aaccgctcct ctaaggatgt gcctcttacc atcaaggatc ctgctgtggg cttcctggag 1560 aaccgctcct ctaaggatgt gcctcttacc atcaaggato ctgctgtggg cttcctggag 1560 acaatctcac ctggctactc cattcacacc tacctgtggc gtcgccagtg a 1611 acaatctcac ctggctactc cattcacacc tacctgtggc gtcgccagtg a 1611

<210> 10 <210> 10 <211> 334 <211> 334 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic construct <223> Synthetic construct

<400> 10 <400> 10 ccctaaaatg ggcaaacatt gcaagcagca aacagcaaac acacagccct ccctgcctgc 60 ccctaaaatg ggcaaacatt gcaagcagca aacagcaaac acacagccct ccctgcctgc 60

tgaccttgga gctggggcag aggtcagaca cctctctggg cccatgccac ctccaactgg 120 tgaccttgga gctggggcag aggtcagaca cctctctggg cccatgccac ctccaactgg 120

acacaggacg ctgtggtttc tgagccaggg ggcgactcag atcccagcca gtggacttag 180 acacaggacg ctgtggtttc tgagccaggg ggcgactcag atcccagcca gtggacttag 180

cccctgtttg ctcctccgat aactggggtg accttggtta atattcacca gcagcctccc 240 cccctgtttg ctcctccgat aactggggtg accttggtta atattcacca gcagcctccc 240

ccgttgcccc tctggatcca ctgcttaaat acggacgagg acagggccct gtctcctcag 300 ccgttgcccc tctggatcca ctgcttaaat acggacgagg acagggccct gtctcctcag 300

cttcaggcac caccactgac ctgggacagt gaat 334 cttcaggcad caccactgad ctgggacagt gaat 334

<210> 11 <210> 11 <211> 117 <211> 117 <212> DNA <212> DNA

<213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic construct <223> Synthetic construct

<400> 11 <400> 11 ccctaaaatg ggcaaacatt gcaagcagca aacagcaaac acacagccct ccctgcctgc 60 ccctaaaatg ggcaaacatt gcaagcagca aacagcaaac acacagccct ccctgcctgc 60

tgaccttgga gctggggcag aggtcagaca cctctctggg cccatgccac ctccaac 117 tgaccttgga gctggggcag aggtcagaca cctctctggg cccatgccac ctccaac 117

<210> 12 <210> 12 <211> 185 <211> 185 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic construct <223> Synthetic construct

<400> 12 <400> 12 gggcgactca gatcccagcc agtggactta gcccctgttt gctcctccga taactggggt 60 gggcgactca gatcccagcc agtggactta gcccctgttt gctcctccga taactggggt 60

gaccttggtt aatattcacc agcagcctcc cccgttgccc ctctggatcc actgcttaaa 120 gaccttggtt aatattcacc agcagcctcc cccgttgccc ctctggatcc actgcttaaa 120

tacggacgag gacagggccc tgtctcctca gcttcaggca ccaccactga cctgggacag 180 tacggacgag gacagggccc tgtctcctca gcttcaggca ccaccactga cctgggacag 180

tgaat 185 tgaat 185

<210> 13 <210> 13 <211> 659 <211> 659 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic construct <223> Synthetic construct

<400> 13 <400> 13 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 60 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagco 60

catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 120 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 120

acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 180 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 180

ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 240 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacato 240

aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 300 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 300

ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 360 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 360

tagtcatcgc tattaccatg gtcgaggtga gccccacgtt ctgcttcact ctccccatct 420 tagtcatcgc tattaccatg gtcgaggtga gccccacgtt ctgcttcact ctccccatct 420

cccccccctc cccaccccca attttgtatt tatttatttt ttaattattt tgtgcagcga 480 cccccccctc cccaccccca attttgtatt tatttatttt ttaattattt tgtgcagcga 480 tgggggcggg gggggggggg gggcgcgcgc caggcggggc ggggcggggc gaggggcggg 540 tgggggcggg gggcgcgcgc caggcggggc ggggcggggc gaggggcggg 540 gcggggcgag gcggagaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc 600 gcggggcgag gcggagaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc 600 cttttatggc gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcg 659 cttttatggc gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcg 659

<210> 14 <210> 14 <211> 747 <211> 747 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic construct <223> Synthetic construct

<400> 14 <400> 14 aggctcagag gcacacagga gtttctgggc tcaccctgcc cccttccaac ccctcagttc 60 aggctcagag gcacacagga gtttctgggc tcaccctgcc cccttccaac ccctcagttc 60

ccatcctcca gcagctgttt gtgtgctgcc tctgaagtcc acactgaaca aacttcagcc 120 ccatcctcca gcagctgttt gtgtgctgcc tctgaagtcc acactgaaca aacttcagcc 120

tactcatgtc cctaaaatgg gcaaacattg caagcagcaa acagcaaaca cacagccctc 180 tactcatgtc cctaaaatgg gcaaacattg caagcagcaa acagcaaaca cacagccctc 180

cctgcctgct gaccttggag ctggggcaga ggtcagagac ctctctgggc ccatgccacc 240 cctgcctgct gaccttggag ctggggcaga ggtcagagac ctctctgggc ccatgccacc 240

tccaacatcc actcgacccc ttggaatttc ggtggagagg agcagaggtt gtcctggcgt 300 tccaacatcc actcgacccc ttggaatttc ggtggagagg agcagaggtt gtcctggcgt 300

ggtttaggta gtgtgagagg ggtacccggg gatcttgcta ccagtggaac agccactaag 360 ggtttaggta gtgtgagagg ggtacccggg gatcttgcta ccagtggaac agccactaag 360

gattctgcag tgagagcaga gggccagcta agtggtactc tcccagagac tgtctgactc 420 gattctgcag tgagagcaga gggccagcta agtggtactc tcccagagad tgtctgactc 420

acgccacccc ctccaccttg gacacaggac gctgtggttt ctgagccagg tacaatgact 480 acgccacccc ctccaccttg gacacaggac gctgtggttt ctgagccagg tacaatgact 480

cctttcggta agtgcagtgg aagctgtaca ctgcccaggc aaagcgtccg ggcagcgtag 540 cctttcggta agtgcagtgg aagctgtaca ctgcccaggo aaagcgtccg ggcagcgtag 540

gcgggcgact cagatcccag ccagtggact tagcccctgt ttgctcctcc gataactggg 600 gcgggcgact cagatcccag ccagtggact tagcccctgt ttgctcctcc gataactggg 600

gtgaccttgg ttaatattca ccagcagcct cccccgttgc ccctctggat ccactgctta 660 gtgaccttgg ttaatattca ccagcagcct cccccgttgc ccctctggat ccactgctta 660

aatacggacg aggacagggc cctgtctcct cagcttcagg caccaccact gacctgggac 720 aatacggacg aggacagggc cctgtctcct cagcttcagg caccaccact gacctgggac 720

agtgaatgat ccccctgatc tgcggcc 747 agtgaatgat ccccctgatc tgcggcc 747

<210> 15 <210> 15 <211> 418 <211> 418 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic construct <223> Synthetic construct

<400> 15 <400> 15 ggatcttgct accagtggaa cagccactaa ggattctgca gtgagagcag agggccagct 60 ggatcttgct accagtggaa cagccactaa ggattctgca gtgagagcag agggccagct 60 aagtggtact ctcccagaga ctgtctgact cacgccaccc cctccacctt ggacacagga 120 aagtggtact ctcccagaga ctgtctgact cacgccacco cctccacctt ggacacagga 120 cgctgtggtt tctgagccag gtacaatgac tcctttcggt aagtgcagtg gaagctgtac 180 cgctgtggtt tctgagccag gtacaatgac tcctttcggt aagtgcagtg gaagctgtac 180 actgcccagg caaagcgtcc gggcagcgta ggcgggcgac tcagatccca gccagtggac 240 actgcccagg caaagcgtcc gggcagcgta ggcgggcgac tcagatccca gccagtggad 240 ttagcccctg tttgctcctc cgataactgg ggtgaccttg gttaatattc accagcagcc 300 ttagcccctg tttgctcctc cgataactgg ggtgaccttg gttaatattc accagcagcc 300 tcccccgttg cccctctgga tccactgctt aaatacggac gaggacaggg ccctgtctcc 360 tcccccgttg cccctctgga tccactgctt aaatacggac gaggacaggg ccctgtctcc 360 tcagcttcag gcaccaccac tgacctggga cagtgaatga tccccctgat ctgcggcc 418 tcagcttcag gcaccaccac tgacctggga cagtgaatga tccccctgat ctgcggcc 418

<210> 16 <210> 16 <211> 321 <211> 321 <212> DNA <212> DNA <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic construct <223> Synthetic construct

<400> 16 <400> 16 aggctcagag gcacacagga gtttctgggc tcaccctgcc cccttccaac ccctcagttc 60 aggctcagag gcacacagga gtttctgggc tcaccctgcc cccttccaac ccctcagttc 60

tccaacatcc actcgacccc ttggaatttc ggtggagagg agcagaggtt gtcctggcgt 300 tccaacatco actcgacccc ttggaatttc ggtggagagg agcagaggtt gtcctggcgt 300

ggtttaggta gtgtgagagg g 321 ggtttaggta gtgtgagagg g 321

<210> 17 <210> 17 <211> 117 <211> 117 <212> DNA <212> DNA <213> Homo sapiens <213> Homo sapiens

<400> 17 <400> 17 atggagtttt caagtccttc cagagaggaa tgtcccaagc ctttgagtag ggtaagcatc 60 atggagtttt caagtccttc cagagaggaa tgtcccaagc ctttgagtag ggtaagcatc 60

atggctggca gcctcacagg attgcttcta cttcaggcag tgtcgtgggc atcaggt 117 atggctggca gcctcacagg attgcttcta cttcaggcag tgtcgtgggc atcaggt 117

<210> 18 <210> 18 <211> 39 <211> 39 <212> PRT <212> PRT <213> Homo sapiens <213> Homo sapiens

<400> 18 <400> 18

Met Glu Phe Ser Ser Pro Ser Arg Glu Glu Cys Pro Lys Pro Leu Ser Met Glu Phe Ser Ser Pro Ser Arg Glu Glu Cys Pro Lys Pro Leu Ser

1 5 10 15 1 5 10 15

Arg Val Ser Ile Met Ala Gly Ser Leu Thr Gly Leu Leu Leu Leu Gln Arg Val Ser Ile Met Ala Gly Ser Leu Thr Gly Leu Leu Leu Leu Gln 20 25 30 20 25 30

Ala Val Ser Trp Ala Ser Gly Ala Val Ser Trp Ala Ser Gly 35 35

<210> 19 <210> 19 <211> 736 <211> 736 <212> PRT <212> PRT <213> adeno‐associated virus 3B <213> adeno-associated virus 3B

<400> 19 <400> 19

Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser Met Ala Ala Asp Gly Tyr Leu Pro Asp Trp Leu Glu Asp Asn Leu Ser 1 5 10 15 1 5 10 15

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Val Pro Gln Pro Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Val Pro Gln Pro 20 25 30 20 25 30

Lys Ala Asn Gln Gln His Gln Asp Asn Arg Arg Gly Leu Val Leu Pro Lys Ala Asn Gln Gln His Gln Asp Asn Arg Arg Gly Leu Val Leu Pro 35 40 45 35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro Gly Tyr Lys Tyr Leu Gly Pro Gly Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 50 55 60

Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp Val Asn Glu Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 70 75 80

Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala Gln Gln Leu Lys Ala Gly Asp Asn Pro Tyr Leu Lys Tyr Asn His Ala 85 90 95 85 90 95

Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly Asp Ala Glu Phe Gln Glu Arg Leu Gln Glu Asp Thr Ser Phe Gly Gly 100 105 110 100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Ile Leu Glu Pro Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Ile Leu Glu Pro 115 120 125 115 120 125

Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg Leu Gly Leu Val Glu Glu Ala Ala Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140 130 135 140

Pro Val Asp Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Val Gly Pro Val Asp Gln Ser Pro Gln Glu Pro Asp Ser Ser Ser Gly Val Gly 145 150 155 160 145 150 155 160

Lys Ser Gly Lys Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr Lys Ser Gly Lys Gln Pro Ala Arg Lys Arg Leu Asn Phe Gly Gln Thr 165 170 175 165 170 175

Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro Gly Asp Ser Glu Ser Val Pro Asp Pro Gln Pro Leu Gly Glu Pro Pro 180 185 190 180 185 190

Ala Ala Pro Thr Ser Leu Gly Ser Asn Thr Met Ala Ser Gly Gly Gly Ala Ala Pro Thr Ser Leu Gly Ser Asn Thr Met Ala Ser Gly Gly Gly 195 200 205 195 200 205

Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser Ala Pro Met Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Asn Ser 210 215 220 210 215 220

Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile Ser Gly Asn Trp His Cys Asp Ser Gln Trp Leu Gly Asp Arg Val Ile 225 230 235 240 225 230 235 240

Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu Thr Thr Ser Thr Arg Thr Trp Ala Leu Pro Thr Tyr Asn Asn His Leu 245 250 255 245 250 255

Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr Tyr Lys Gln Ile Ser Ser Gln Ser Gly Ala Ser Asn Asp Asn His Tyr 260 265 270 260 265 270

Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Phe His 275 280 285 275 280 285

Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Asn Trp 290 295 300 290 295 300

Gly Phe Arg Pro Lys Lys Leu Ser Phe Lys Leu Phe Asn Ile Gln Val Gly Phe Arg Pro Lys Lys Leu Ser Phe Lys Leu Phe Asn Ile Gln Val 305 310 315 320 305 310 315 320

Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu Lys Glu Val Thr Gln Asn Asp Gly Thr Thr Thr Ile Ala Asn Asn Leu 325 330 335 325 330 335

Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr Thr Ser Thr Val Gln Val Phe Thr Asp Ser Glu Tyr Gln Leu Pro Tyr 340 345 350 340 345 350

Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp Val Leu Gly Ser Ala His Gln Gly Cys Leu Pro Pro Phe Pro Ala Asp 355 360 365 355 360 365

Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser Val Phe Met Val Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asn Gly Ser 370 375 380 370 375 380

Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Pro Ser 385 390 395 400 385 390 395 400

Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Thr Phe Glu Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Thr Phe Glu 405 410 415 405 410 415

Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg Asp Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Asp Arg 420 425 430 420 425 430

Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg Thr Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Asn Arg Thr 435 440 445 435 440 445

Gln Gly Thr Thr Ser Gly Thr Thr Asn Gln Ser Arg Leu Leu Phe Ser Gln Gly Thr Thr Ser Gly Thr Thr Asn Gln Ser Arg Leu Leu Phe Ser 450 455 460 450 455 460

Gln Ala Gly Pro Gln Ser Met Ser Leu Gln Ala Arg Asn Trp Leu Pro Gln Ala Gly Pro Gln Ser Met Ser Leu Gln Ala Arg Asn Trp Leu Pro 465 470 475 480 465 470 475 480

Gly Pro Cys Tyr Arg Gln Gln Arg Leu Ser Lys Thr Ala Asn Asp Asn Gly Pro Cys Tyr Arg Gln Gln Arg Leu Ser Lys Thr Ala Asn Asp Asn 485 490 495 485 490 495

Asn Asn Ser Asn Phe Pro Trp Thr Ala Ala Ser Lys Tyr His Leu Asn Asn Asn Ser Asn Phe Pro Trp Thr Ala Ala Ser Lys Tyr His Leu Asn 500 505 510 500 505 510

Gly Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys Gly Arg Asp Ser Leu Val Asn Pro Gly Pro Ala Met Ala Ser His Lys 515 520 525 515 520 525

Asp Asp Glu Glu Lys Phe Phe Pro Met His Gly Asn Leu Ile Phe Gly Asp Asp Glu Glu Lys Phe Phe Pro Met His Gly Asn Leu Ile Phe Gly 530 535 540 530 535 540

Lys Glu Gly Thr Thr Ala Ser Asn Ala Glu Leu Asp Asn Val Met Ile Lys Glu Gly Thr Thr Ala Ser Asn Ala Glu Leu Asp Asn Val Met Ile 545 550 555 560 545 550 555 560

Thr Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln Thr Asp Glu Glu Glu Ile Arg Thr Thr Asn Pro Val Ala Thr Glu Gln 565 570 575 565 570 575

Tyr Gly Thr Val Ala Asn Asn Leu Gln Ser Ser Asn Thr Ala Pro Thr Tyr Gly Thr Val Ala Asn Asn Leu Gln Ser Ser Asn Thr Ala Pro Thr 580 585 590 580 585 590

Thr Arg Thr Val Asn Asp Gln Gly Ala Leu Pro Gly Met Val Trp Gln Thr Arg Thr Val Asn Asp Gln Gly Ala Leu Pro Gly Met Val Trp Gln 595 600 605 595 600 605

Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His Asp Arg Asp Val Tyr Leu Gln Gly Pro Ile Trp Ala Lys Ile Pro His 610 615 620 610 615 620

Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu Thr Asp Gly His Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Leu 625 630 635 640 625 630 635 640

Lys His Pro Pro Pro Gln Ile Met Ile Lys Asn Thr Pro Val Pro Ala Lys His Pro Pro Pro Gln Ile Met Ile Lys Asn Thr Pro Val Pro Ala 645 650 655 645 650 655

Asn Pro Pro Thr Thr Phe Ser Pro Ala Lys Phe Ala Ser Phe Ile Thr Asn Pro Pro Thr Thr Phe Ser Pro Ala Lys Phe Ala Ser Phe Ile Thr 660 665 670 660 665 670

Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln Gln Tyr Ser Thr Gly Gln Val Ser Val Glu Ile Glu Trp Glu Leu Gln 675 680 685 675 680 685

Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn Lys Glu Asn Ser Lys Arg Trp Asn Pro Glu Ile Gln Tyr Thr Ser Asn 690 695 700 690 695 700

Tyr Asn Lys Ser Val Asn Val Asp Phe Thr Val Asp Thr Asn Gly Val Tyr Asn Lys Ser Val Asn Val Asp Phe Thr Val Asp Thr Asn Gly Val 705 710 715 720 705 710 715 720

Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Asn Leu 725 730 735 725 730 735

<210> 20 <210> 20 <211> 736 <211> 736 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic capsid protein <223> Synthetic capsid protein

<400> 20 <400> 20

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Lys Pro Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Lys Pro 20 25 30 20 25 30

Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro Lys Ala Asn Gln Gln Lys Gln Asp Asp Gly Arg Gly Leu Val Leu Pro 35 40 45 35 40 45

Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro Gly Tyr Lys Tyr Leu Gly Pro Phe Asn Gly Leu Asp Lys Gly Glu Pro 50 55 60 50 55 60

Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp Val Asn Ala Ala Asp Ala Ala Ala Leu Glu His Asp Lys Ala Tyr Asp 65 70 75 80 70 75 80

Gln Gln Leu Gln Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala Gln Gln Leu Gln Ala Gly Asp Asn Pro Tyr Leu Arg Tyr Asn His Ala 85 90 95 85 90 95

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Val Leu Glu Pro 115 120 125 115 120 125

Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg Leu Gly Leu Val Glu Glu Gly Ala Lys Thr Ala Pro Gly Lys Lys Arg 130 135 140 130 135 140

<210> 21 <210> 21 <211> 736 <211> 736 <212> PRT <212> PRT <213> adeno‐associated virus 9 <213> adeno-associated virus 9

<400> 21 <400> 21

Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln Pro Glu Gly Ile Arg Glu Trp Trp Ala Leu Lys Pro Gly Ala Pro Gln Pro 20 25 30 20 25 30

Lys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro Lys Ala Asn Gln Gln His Gln Asp Asn Ala Arg Gly Leu Val Leu Pro 35 40 45 35 40 45

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly 100 105 110 100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro

115 120 125 115 120 125

Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ala Gly Ile Gly Pro Val Glu Gln Ser Pro Gln Glu Pro Asp Ser Ser Ala Gly Ile Gly 145 150 155 160 145 150 155 160

Lys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr Lys Ser Gly Ala Gln Pro Ala Lys Lys Arg Leu Asn Phe Gly Gln Thr 165 170 175 165 170 175

Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro Gly Asp Thr Glu Ser Val Pro Asp Pro Gln Pro Ile Gly Glu Pro Pro 180 185 190 180 185 190

Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly Ala Ala Pro Ser Gly Val Gly Ser Leu Thr Met Ala Ser Gly Gly Gly 195 200 205 195 200 205

Ala Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser Ser Ala Pro Val Ala Asp Asn Asn Glu Gly Ala Asp Gly Val Gly Ser Ser 210 215 220 210 215 220

Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp Asn Tyr Lys Gln Ile Ser Asn Ser Thr Ser Gly Gly Ser Ser Asn Asp Asn 260 265 270 260 265 270

Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg Ala Tyr Phe Gly Tyr Ser Thr Pro Trp Gly Tyr Phe Asp Phe Asn Arg 275 280 285 275 280 285

Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn Phe His Cys His Phe Ser Pro Arg Asp Trp Gln Arg Leu Ile Asn Asn 290 295 300 290 295 300

Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile Asn Trp Gly Phe Arg Pro Lys Arg Leu Asn Phe Lys Leu Phe Asn Ile 305 310 315 320 305 310 315 320

Gln Val Lys Glu Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala Asn Gln Val Lys Glu Val Thr Asp Asn Asn Gly Val Lys Thr Ile Ala Asn 325 330 335 325 330 335

Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln Leu Asn Leu Thr Ser Thr Val Gln Val Phe Thr Asp Ser Asp Tyr Gln Leu 340 345 350 340 345 350

Pro Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe Pro Pro Tyr Val Leu Gly Ser Ala His Glu Gly Cys Leu Pro Pro Phe Pro 355 360 365 355 360 365

Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asp Ala Asp Val Phe Met Ile Pro Gln Tyr Gly Tyr Leu Thr Leu Asn Asp 370 375 380 370 375 380

Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe Gly Ser Gln Ala Val Gly Arg Ser Ser Phe Tyr Cys Leu Glu Tyr Phe 385 390 395 400 385 390 395 400

Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Glu Pro Ser Gln Met Leu Arg Thr Gly Asn Asn Phe Gln Phe Ser Tyr Glu 405 410 415 405 410 415

Phe Glu Asn Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu Phe Glu Asn Val Pro Phe His Ser Ser Tyr Ala His Ser Gln Ser Leu 420 425 430 420 425 430

Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser Asp Arg Leu Met Asn Pro Leu Ile Asp Gln Tyr Leu Tyr Tyr Leu Ser 435 440 445 435 440 445

Lys Thr Ile Asn Gly Ser Gly Gln Asn Gln Gln Thr Leu Lys Phe Ser Lys Thr Ile Asn Gly Ser Gly Gln Asn Gln Gln Thr Leu Lys Phe Ser 450 455 460 450 455 460

Val Ala Gly Pro Ser Asn Met Ala Val Gln Gly Arg Asn Tyr Ile Pro Val Ala Gly Pro Ser Asn Met Ala Val Gln Gly Arg Asn Tyr Ile Pro 465 470 475 480 465 470 475 480

Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Val Thr Gln Asn Gly Pro Ser Tyr Arg Gln Gln Arg Val Ser Thr Thr Val Thr Gln Asn 485 490 495 485 490 495

Asn Asn Ser Glu Phe Ala Trp Pro Gly Ala Ser Ser Trp Ala Leu Asn Asn Asn Ser Glu Phe Ala Trp Pro Gly Ala Ser Ser Trp Ala Leu Asn 500 505 510 500 505 510

Gly Arg Asn Ser Leu Met Asn Pro Gly Pro Ala Met Ala Ser His Lys Gly Arg Asn Ser Leu Met Asn Pro Gly Pro Ala Met Ala Ser His Lys 515 520 525 515 520 525

Glu Gly Glu Asp Arg Phe Phe Pro Leu Ser Gly Ser Leu Ile Phe Gly Glu Gly Glu Asp Arg Phe Phe Pro Leu Ser Gly Ser Leu Ile Phe Gly 530 535 540 530 535 540

Lys Gln Gly Thr Gly Arg Asp Asn Val Asp Ala Asp Lys Val Met Ile Lys Gln Gly Thr Gly Arg Asp Asn Val Asp Ala Asp Lys Val Met Ile

545 550 555 560 545 550 555 560

Thr Asn Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Ser Thr Asn Glu Glu Glu Ile Lys Thr Thr Asn Pro Val Ala Thr Glu Ser 565 570 575 565 570 575

Tyr Gly Gln Val Ala Thr Asn His Gln Ser Ala Gln Ala Gln Ala Gln Tyr Gly Gln Val Ala Thr Asn His Gln Ser Ala Gln Ala Gln Ala Gln 580 585 590 580 585 590

Thr Gly Trp Val Gln Asn Gln Gly Ile Leu Pro Gly Met Val Trp Gln Thr Gly Trp Val Gln Asn Gln Gly Ile Leu Pro Gly Met Val Trp Gln 595 600 605 595 600 605

Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Met Thr Asp Gly Asn Phe His Pro Ser Pro Leu Met Gly Gly Phe Gly Met 625 630 635 640 625 630 635 640

Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala Lys His Pro Pro Pro Gln Ile Leu Ile Lys Asn Thr Pro Val Pro Ala 645 650 655 645 650 655

Asp Pro Pro Thr Ala Phe Asn Lys Asp Lys Leu Asn Ser Phe Ile Thr Asp Pro Pro Thr Ala Phe Asn Lys Asp Lys Leu Asn Ser Phe Ile Thr 660 665 670 660 665 670

Tyr Tyr Lys Ser Asn Asn Val Glu Phe Ala Val Asn Thr Glu Gly Val Tyr Tyr Lys Ser Asn Asn Val Glu Phe Ala Val Asn Thr Glu Gly Val 705 710 715 720 705 710 715 720

<210> 22 <210> 22 <211> 66 <211> 66 <212> DNA <212> DNA <213> Simian virus 40 <213> Simian virus 40

<400> 22 4001 22 gtaaatataa aatttttaag tgtataatgt gttaaactac tgattctaat tgtttctctc 60 gtaaatataa aatttttaag tgtataatgt gttaaactac tgattctaat tgtttctctc 60 ttttag 66 ttttag 66

<210> 23 <210> 23 <211> 208 <211> 208 <212> DNA <212> DNA <213> Bos taurus <213> Bos taurus

<400> 23 <400> 23 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc 60 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgacco 60

tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc 120 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgto 120

tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 180 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 180

gggaagacaa tagcaggcat gctgggga 208 gggaagacaa tagcaggcat gctgggga 208

<210> 24 <210> 24 <211> 736 <211> 736 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence

<220> <220> <223> Synthetic capsid protein <223> Synthetic capsid protein

<400> 24 <400> 24

Glu Gly Ile Arg Glu Trp Trp Ala Leu Gln Pro Gly Ala Pro Lys Pro Glu Gly Ile Arg Glu Trp Trp Ala Leu Gln Pro Gly Ala Pro Lys Pro 20 25 30 20 25 30

Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly Asp Ala Glu Phe Gln Glu Arg Leu Lys Glu Asp Thr Ser Phe Gly Gly

100 105 110 100 105 110

Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro Asn Leu Gly Arg Ala Val Phe Gln Ala Lys Lys Arg Leu Leu Glu Pro 115 120 125 115 120 125

Asp Asp Glu Glu Lys Phe Phe Pro Met His Gly Asn Leu Ile Phe Gly Asp Asp Glu Glu Lys Phe Phe Pro Met His Gly Asn Leu Ile Phe Gly

530 535 540 530 535 540

Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu Tyr Ser Glu Pro Arg Pro Ile Gly Thr Arg Tyr Leu Thr Arg Pro Leu 725 730 735 725 730 735

<210> 25 <210> 25 <211> 536 <211> 536 <212> PRT <212> PRT

<213> Homo sapiens <213> Homo sapiens

<400> 25 <400> 25

Met Glu Phe Ser Ser Pro Ser Arg Glu Glu Cys Pro Lys Pro Leu Ser Met Glu Phe Ser Ser Pro Ser Arg Glu Glu Cys Pro Lys Pro Leu Ser 1 5 10 15 1 5 10 15

Ala Val Ser Trp Ala Ser Gly Ala Arg Pro Cys Ile Pro Lys Ser Phe Ala Val Ser Trp Ala Ser Gly Ala Arg Pro Cys Ile Pro Lys Ser Phe 35 40 45 35 40 45

Gly Tyr Ser Ser Val Val Cys Val Cys Asn Ala Thr Tyr Cys Asp Ser Gly Tyr Ser Ser Val Val Cys Val Cys Asn Ala Thr Tyr Cys Asp Ser 50 55 60 50 55 60

Phe Asp Pro Pro Thr Phe Pro Ala Leu Gly Thr Phe Ser Arg Tyr Glu Phe Asp Pro Pro Thr Phe Pro Ala Leu Gly Thr Phe Ser Arg Tyr Glu 65 70 75 80 70 75 80

Ser Thr Arg Ser Gly Arg Arg Met Glu Leu Ser Met Gly Pro Ile Gln Ser Thr Arg Ser Gly Arg Arg Met Glu Leu Ser Met Gly Pro Ile Gln 85 90 95 85 90 95

Ala Asn His Thr Gly Thr Gly Leu Leu Leu Thr Leu Gln Pro Glu Gln Ala Asn His Thr Gly Thr Gly Leu Leu Leu Thr Leu Gln Pro Glu Gln 100 105 110 100 105 110

Lys Phe Gln Lys Val Lys Gly Phe Gly Gly Ala Met Thr Asp Ala Ala Lys Phe Gln Lys Val Lys Gly Phe Gly Gly Ala Met Thr Asp Ala Ala 115 120 125 115 120 125

Ala Leu Asn Ile Leu Ala Leu Ser Pro Pro Ala Gln Asn Leu Leu Leu Ala Leu Asn Ile Leu Ala Leu Ser Pro Pro Ala Gln Asn Leu Leu Leu 130 135 140 130 135 140

Lys Ser Tyr Phe Ser Glu Glu Gly Ile Gly Tyr Asn Ile Ile Arg Val Lys Ser Tyr Phe Ser Glu Glu Gly Ile Gly Tyr Asn Ile Ile Arg Val 145 150 155 160 145 150 155 160

Pro Met Ala Ser Cys Asp Phe Ser Ile Arg Thr Tyr Thr Tyr Ala Asp Pro Met Ala Ser Cys Asp Phe Ser Ile Arg Thr Tyr Thr Tyr Ala Asp 165 170 175 165 170 175

Thr Pro Asp Asp Phe Gln Leu His Asn Phe Ser Leu Pro Glu Glu Asp Thr Pro Asp Asp Phe Gln Leu His Asn Phe Ser Leu Pro Glu Glu Asp 180 185 190 180 185 190

Thr Lys Leu Lys Ile Pro Leu Ile His Arg Ala Leu Gln Leu Ala Gln Thr Lys Leu Lys Ile Pro Leu Ile His Arg Ala Leu Gln Leu Ala Gln 195 200 205 195 200 205

Arg Pro Val Ser Leu Leu Ala Ser Pro Trp Thr Ser Pro Thr Trp Leu Arg Pro Val Ser Leu Leu Ala Ser Pro Trp Thr Ser Pro Thr Trp Leu 210 215 220 210 215 220

Lys Thr Asn Gly Ala Val Asn Gly Lys Gly Ser Leu Lys Gly Gln Pro Lys Thr Asn Gly Ala Val Asn Gly Lys Gly Ser Leu Lys Gly Gln Pro 225 230 235 240 225 230 235 240

Gly Asp Ile Tyr His Gln Thr Trp Ala Arg Tyr Phe Val Lys Phe Leu Gly Asp Ile Tyr His Gln Thr Trp Ala Arg Tyr Phe Val Lys Phe Leu 245 250 255 245 250 255

Asp Ala Tyr Ala Glu His Lys Leu Gln Phe Trp Ala Val Thr Ala Glu Asp Ala Tyr Ala Glu His Lys Leu Gln Phe Trp Ala Val Thr Ala Glu 260 265 270 260 265 270

Asn Glu Pro Ser Ala Gly Leu Leu Ser Gly Tyr Pro Phe Gln Cys Leu Asn Glu Pro Ser Ala Gly Leu Leu Ser Gly Tyr Pro Phe Gln Cys Leu 275 280 285 275 280 285

Gly Phe Thr Pro Glu His Gln Arg Asp Phe Ile Ala Arg Asp Leu Gly Gly Phe Thr Pro Glu His Gln Arg Asp Phe Ile Ala Arg Asp Leu Gly 290 295 300 290 295 300

Pro Thr Leu Ala Asn Ser Thr His His Asn Val Arg Leu Leu Met Leu Pro Thr Leu Ala Asn Ser Thr His His Asn Val Arg Leu Leu Met Leu 305 310 315 320 305 310 315 320

Asp Asp Gln Arg Leu Leu Leu Pro His Trp Ala Lys Val Val Leu Thr Asp Asp Gln Arg Leu Leu Leu Pro His Trp Ala Lys Val Val Leu Thr 325 330 335 325 330 335

Asp Pro Glu Ala Ala Lys Tyr Val His Gly Ile Ala Val His Trp Tyr Asp Pro Glu Ala Ala Lys Tyr Val His Gly Ile Ala Val His Trp Tyr 340 345 350 340 345 350

Leu Asp Phe Leu Ala Pro Ala Lys Ala Thr Leu Gly Glu Thr His Arg Leu Asp Phe Leu Ala Pro Ala Lys Ala Thr Leu Gly Glu Thr His Arg 355 360 365 355 360 365

Leu Phe Pro Asn Thr Met Leu Phe Ala Ser Glu Ala Cys Val Gly Ser Leu Phe Pro Asn Thr Met Leu Phe Ala Ser Glu Ala Cys Val Gly Ser 370 375 380 370 375 380

Lys Phe Trp Glu Gln Ser Val Arg Leu Gly Ser Trp Asp Arg Gly Met Lys Phe Trp Glu Gln Ser Val Arg Leu Gly Ser Trp Asp Arg Gly Met 385 390 395 400 385 390 395 400

Gln Tyr Ser His Ser Ile Ile Thr Asn Leu Leu Tyr His Val Val Gly Gln Tyr Ser His Ser Ile Ile Thr Asn Leu Leu Tyr His Val Val Gly 405 410 415 405 410 415

Trp Thr Asp Trp Asn Leu Ala Leu Asn Pro Glu Gly Gly Pro Asn Trp Trp Thr Asp Trp Asn Leu Ala Leu Asn Pro Glu Gly Gly Pro Asn Trp 420 425 430 420 425 430

Val Arg Asn Phe Val Asp Ser Pro Ile Ile Val Asp Ile Thr Lys Asp Val Arg Asn Phe Val Asp Ser Pro Ile Ile Val Asp Ile Thr Lys Asp 435 440 445 435 440 445

Thr Phe Tyr Lys Gln Pro Met Phe Tyr His Leu Gly His Phe Ser Lys Thr Phe Tyr Lys Gln Pro Met Phe Tyr His Leu Gly His Phe Ser Lys 450 455 460 450 455 460

Phe Ile Pro Glu Gly Ser Gln Arg Val Gly Leu Val Ala Ser Gln Lys Phe Ile Pro Glu Gly Ser Gln Arg Val Gly Leu Val Ala Ser Gln Lys 465 470 475 480 465 470 475 480

Asn Asp Leu Asp Ala Val Ala Leu Met His Pro Asp Gly Ser Ala Val Asn Asp Leu Asp Ala Val Ala Leu Met His Pro Asp Gly Ser Ala Val 485 490 495 485 490 495

Val Val Val Leu Asn Arg Ser Ser Lys Asp Val Pro Leu Thr Ile Lys Val Val Val Leu Asn Arg Ser Ser Lys Asp Val Pro Leu Thr Ile Lys 500 505 510 500 505 510

Asp Pro Ala Val Gly Phe Leu Glu Thr Ile Ser Pro Gly Tyr Ser Ile Asp Pro Ala Val Gly Phe Leu Glu Thr Ile Ser Pro Gly Tyr Ser Ile 515 520 525 515 520 525

His Thr Tyr Leu Trp Arg Arg Gln His Thr Tyr Leu Trp Arg Arg Gln 530 535 530 535

Claims

Claims 24 Jul 2025

1. A polynucleotide comprising a GBA nucleotide sequence, wherein the GBA nucleotide sequence encodes a β-Glucocerebrosidase (GCase) protein and wherein the GBA nucleotide sequence comprises a sequence that is: (i) 100% identical to SEQ ID NO: 1 or SEQ ID NO: 5; (ii) at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, 2020217894

or at least 99.8% identical to SEQ ID NO: 1 or SEQ ID NO: 5, wherein the GCase encoded by the GBA nucleotide sequence has GCase activity and expresses in human liver cells at higher levels compared to a GCase encoded by a wild type GBA nucleotide sequence in an otherwise identical reference polynucleotide; and/or (iii) a variant of SEQ ID NO: 1 or SEQ ID NO: 5 encoding a GCase protein having GCase activity, wherein the variant is identical to SEQ ID NO: 1 or SEQ ID NO: 5 respectively except that it comprises nucleotide substitutions such that the GCase protein has 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, or up to 10 amino acid substitutions relative to the wild type GCase amino acid sequence of SEQ ID NO: 25, further wherein the GCase encoded by the GBA nucleotide expresses in human liver cells at higher levels compared to a GCase encoded by a wild type GBA nucleotide sequence in an otherwise identical reference polynucleotide.

2. The polynucleotide of claim 1, wherein: (a) the variant is a variant of SEQ ID NO: 1 and the variant of SEQ ID NO: 1: (i) is identical to SEQ ID NO: 1 except that it comprises nucleotide substitutions such that the GCase protein has 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, or up to 10 amino acid substitutions relative to the wild type GCase amino acid sequence of SEQ ID NO: 25; (ii) has 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 20, or up to 30 nucleotide substitutions relative to the sequence of SEQ ID NO: 1; (iii) has 1, up to 2, up to 3, up to 4, up to 5, or up to 6 nucleotide substitutions relative to the sequence of SEQ ID NO: 1; (iv) has up to 4 nucleotide substitutions relative to the sequence of SEQ ID NO: 1 and/or encodes a GCase protein having up to 3 amino acid substitutions relative to the wild type amino acid GCase sequence of SEQ ID NO: 25;

(v) has up to 3 nucleotide substitutions relative to the sequence of SEQ ID NO: 1 24 Jul 2025

and/or encodes a GCase protein having up to 2 amino acid substitutions relative to the wild type GCase amino acid sequence of SEQ ID NO: 25; and/or (vi) has 1 nucleotide substitution relative to the sequence of SEQ ID NO: 1 and/or encodes a GCase protein having up to 1 amino acid substitution relative to the wild type GCase amino acid sequence of SEQ ID NO: 25; or 2020217894

(b) the variant is a variant of SEQ ID NO: 5 and the variant of SEQ ID NO: 5: (i) is identical to SEQ ID NO: 5 except that it comprises nucleotide substitutions such that the GCase protein has 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, or up to 10 amino acid substitutions relative to the wild type GCase amino acid sequence of SEQ ID NO: 25; (ii) has 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 20, or up to 30 nucleotide substitutions relative to the sequence of SEQ ID NO: 5; (iii) has 1, up to 2, up to 3, up to 4, up to 5, or up to 6 nucleotide substitutions relative to the sequence of SEQ ID NO: 5; (iv) has up to 4 nucleotide substitutions relative to the sequence of SEQ ID NO: 5 and/or encodes a GCase protein having up to 3 amino acid substitutions relative to the wild type amino acid GCase sequence of SEQ ID NO: 25; (v) has up to 3 nucleotide substitutions relative to the sequence of SEQ ID NO: 5 and/or encodes a GCase protein having up to 2 amino acid substitutions relative to the wild type GCase amino acid sequence of SEQ ID NO: 25; and/or (vi) has 1 nucleotide substitution relative to the sequence of SEQ ID NO: 5 and/or encodes a GCase protein having up to 1 amino acid substitution relative to the wild type GCase amino acid sequence of SEQ ID NO: 25.

3. The polynucleotide of claim 1 or claim 2, wherein the variant has: (i) up to 3 amino acid substitutions relative to the wild type GCase amino acid sequence of SEQ ID NO: 25; (ii) up to 2 amino acid substitutions relative to the wild type GCase amino acid sequence of SEQ ID NO: 25; and/or (iii) up to 1 amino acid substitution relative to the wild type GCase amino acid sequence of SEQ ID NO: 25.

4. The polynucleotide of any one of the preceding claims, wherein the GCase encoded by 24 Jul 2025

the GBA nucleotide sequence expresses in human liver cells at higher levels compared to a GCase encoded by a wild type GBA nucleotide sequence in an otherwise identical reference polynucleotide and wherein: (a) the GBA nucleotide sequence is codon-optimised for expression in human liver cells; and/or (b) the GBA nucleotide sequence comprises a reduced number of CpGs compared to a 2020217894

wild type GBA nucleotide sequence; and/or (c) the GBA nucleotide sequence comprises a reduced number of CpGs compared to a wild type GBA nucleotide sequence, wherein the GBA nucleotide sequence comprises (i) less than 40 CpGs; (ii) less than 20 CpGs; (iii) less than 18 CpGs; (iv) less than 10 CpGs; (v) less than 5 CpGs per 100 nucleotides; (vi) less than 4 CpGs per 100 nucleotides; (vii) less than 3 CpGs per 100 nucleotides; (viii) less than 2 CpGs per 100 nucleotides; or (ix) the GBA nucleotide sequence is CpG-free; and/or (d) the wild type GBA nucleotide sequence is SEQ ID NO: 9.

5. The polynucleotide of any one of the preceding claims, wherein: (I) the polynucleotide further comprises a transcription regulatory element; (II) the polynucleotide further comprises a transcription regulatory element wherein the transcription regulatory element comprises a liver-specific promoter and/or an enhancer; (III) the polynucleotide further comprises a transcription regulatory element wherein: (a)(i) the transcription regulatory element comprises an A1AT promoter or a fragment of an A1AT promoter; (ii) the transcription regulatory element comprises an A1AT promoter or a fragment of an A1AT promoter, wherein the A1AT promoter or the fragment of an A1AT promoter is at least 100, at least 120, at least 150, at least 180, less than 255, between 100 and 255, between 150 and 225, between 150 and 300, or between 180 and 255 nucleotides in length;

(iii) the transcription regulatory element comprises an A1AT promoter or a fragment 24 Jul 2025

of an A1AT promoter, wherein the A1AT promoter or the fragment of an A1AT promoter is at least 200, at least 250, at least 300, less than 500, between 200 and 500, between 250 and 500, between 350 and 450, or 418 nucleotides in length; and/or (iv) the transcription regulatory element comprises an A1AT promoter or a fragment of an A1AT promoter, wherein the polynucleotide comprises a promoter that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, 2020217894

at least 99.8%, or 100% identical to SEQ ID NO: 15 or SEQ ID NO: 12; and/or

(b)(i) the enhancer is an HCR enhancer or a fragment of an HCR enhancer ; (ii) the enhancer is an HCR enhancer or a fragment of an HCR enhancer, wherein the HCR enhancer or the fragment of an HCR enhancer is a fragment of at least 80, at least 90, at least 100, less than 192, between 80 and 192, between 90 and 192, between 100 and 250, or between 117 and 192 nucleotides in length; (iii) the enhancer is an HCR enhancer or a fragment of an HCR enhancer, wherein the HCR enhancer or the fragment of an HCR enhancer is a fragment of at least 150, at least 190, at least 230, less than 400, between 150 and 400, between 190 and 370, between 230 and 340, between 250 and 340, or 321 nucleotides in length; or (iv) the enhancer is an HCR enhancer or a fragment of an HCR enhancer, wherein the polynucleotide comprises an enhancer that is at least 80%, at least 85%, at least 90%, at least 95% at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 16 or SEQ ID NO: 11; or

(IV) the transcription regulatory element is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO: 14 or 10.

6. The polynucleotide of any one of the preceding claims, wherein: (a) the GCase encoded by the GBA nucleotide sequence expresses in human liver cells at least 1.1x, at least 1.2x, at least 1.3x, at least 1.4x, or at least 1.5x higher compared to a GCase encoded by a wild type GBA nucleotide sequence in an otherwise identical reference polynucleotide; (b) the GCase of claim 6(a) wherein the reference polynucleotide comprises a wild type GBA nucleotide sequence of SEQ ID NO: 9; or

(c) the GCase of claim 6(a) and (b) wherein the reference polynucleotide comprises a 24 Jul 2025

promoter of SEQ ID NO: 13.

7. A viral particle comprising a recombinant genome comprising the polynucleotide of any one of the preceding claims.

8. The viral particle of claim 7, wherein: 2020217894

(a)(i) the viral particle is an AAV, adenoviral, or lentiviral viral particle, or (ii) the viral particle is an AAV viral particle; and/or (b)(i) the viral particle comprises a liver-tropic or CNS-tropic capsid; (ii) the viral particle comprises a liver-tropic or CNS-tropic capsid wherein the liver- tropic capsid comprises a sequence at least 98%, at least 99%, or at least 99.5% identical to a fragment of at least 600, at least 650, at least 700, between 600 and 736, between 650 and 736 or between 700 and 736 amino acids of SEQ ID NO: 19, 20 or 24; (iii) the viral particle comprises a liver-tropic or CNS-tropic capsid wherein the liver- tropic capsid comprises a sequence at least 99% identical to SEQ ID NO: 19 or 20; (iv) the viral particle comprises a liver-tropic or CNS-tropic capsid wherein the CNS- tropic capsid comprises a sequence at least 98%, at least 99%, or at least 99.5% identical to a fragment of at least 600, at least 650, at least 700, between 600 and 736, between 650 and 736 or between 700 and 736 amino acids of SEQ ID NO: 21, or (v) the viral particle comprises a liver-tropic or CNS-tropic capsid wherein the CNS- tropic capsid comprises a sequence at least 99% identical to SEQ ID NO: 21; and/or (c)(i) the recombinant genome further comprises: i) AAV2 ITRs; ii) a poly A sequence; and/or iii) an intron; or (ii) the recombinant genome further comprises: i) AAV2 ITRs; ii) a poly A sequence; and/or iii) an intron; wherein the recombinant genome is single-stranded; and/or (d) on transduction into Huh-7 cells,

(i) the viral particle expresses GCase such that the GCase activity in the 24 Jul 2025

transduced cell is greater than the activity of GCase in a cell transduced with an otherwise identical viral particle comprising a GBA nucleotide sequence of SEQ ID NO: 9; (ii) the viral particle expresses GCase such that the GCase activity in the transduced cell is at least 2x, at least 3x, at least 4x, at least 5x, at least 10x, or at least 20x greater than the activity of GCase in a cell transduced with an otherwise 2020217894

identical viral particle comprising a GBA nucleotide sequence of SEQ ID NO: 9; (iii) the viral particle expresses GCase such that the GCase activity in the transduced cell is greater than the activity of GCase in a cell transduced with an otherwise identical viral particle comprising a GBA nucleotide sequence of SEQ ID NO: 9, wherein the activity is measured using a fluorometric substrate which is specific for GCase; or (iv) the viral particle expresses GCase such that the GCase activity in the transduced cell is at least 2x, at least 3x, at least 4x, at least 5x, at least 10x, or at least 20x greater than the activity of GCase in a cell transduced with an otherwise identical viral particle comprising a GBA nucleotide sequence of SEQ ID NO: 9, wherein the activity is measured using a fluorometric substrate which is specific for GCase.

9. A composition comprising the polynucleotide of any one of claims 1 to 6 or the viral particle of claim 7 or claim 8; and a pharmaceutically acceptable excipient.

10. A method of treatment comprising administering an effective amount of the polynucleotide of any one of claims 1 to 6, the viral particle of claim 7 or claim 8 or the composition of claim 9, wherein the method is (a) a method of treating Gaucher disease; (b) a method of treating Gaucher disease wherein the Gaucher disease is type I, II or III; and/or (c) a method of treating Gaucher disease wherein the patient has antibodies or inhibitors to a recombinant GCase with which the patient has previously been treated as part of an enzyme replacement therapy.

11. A method of expressing a GBA nucleotide sequence and (i) achieving a stable GCase 24 Jul 2025

activity in a subject and/or (ii) providing greater GCase bioavailability in a subject compared to the bioavailability from GCase enzyme replacement therapy, wherein the bioavailability is measured over a period of 2 weeks from administration, the method comprising administering to the subject an effective amount of the polynucleotide of any one of claims 1 to 6, the viral particle of claim 7 or claim 8 or the composition of claim 9. 2020217894

12. The method of claim 11, wherein achieving a stable GCase activity and/or providing greater GCase bioavailability leads to the treatment of a disease in the subject.

13. The method of claim 11 or claim 12, wherein: (i) the GCase activity and/or bioavailability is measured using a fluorometric substrate which is specific for GCase; (ii) the GCase activity is measured in the serum or plasma of the subject; (iii) the GCase activity is measured in the macrophages of the subject; (iv) the GCase activity is stable at a level of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, or at least 9 µmol/h/ml in the subject; (v) the GCase activity is stable at a level of at least 3 µmol/h/ml in the subject; (vi) the GCase activity is stable at a level of at least 5 µmol/h/ml in the subject; (vii) the GCase activity is stable at a level of at least 9 µmol/h/ml in the subject; (viii) the method comprises administering an effective dose of the polynucleotide, viral particle or composition to the subject; (ix) the stable GCase activity is a GCase activity of at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% relative to the GCase activity of a healthy subject; (x) the stable GCase activity is a GCase activity of between 10% and 100%, between 20% and 90%, between 30% and 70%, between 40% and 70%, or between 50% and 70% relative to the GCase activity of a healthy subject; (xi) the stable GCase activity is stable for at least 5 weeks from administration; (xii) the stable GCase activity is stable for at least 10 weeks from administration; (xiii) the stable GCase activity is stable for at least 15 weeks from administration; (xiv) the stable GCase activity is stable for at least 20 weeks from administration; (xv) the stable GCase activity is stable for at least 25 weeks from administration; (xvi) the stable GCase activity is stable for at least 30 weeks from administration;

(xvii) the stable GCase activity is stable for at least 35 weeks from administration; 24 Jul 2025

(xviii) the stable GCase activity is stable for at least 40 weeks after administration; (xix) the method achieves a greater GCase activity in the liver, spleen, and/or bone marrow of the subject at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, or at least 35 weeks after administration when compared to the activity measured in a subject administered an effective dose of a GCase enzyme replacement therapy, when measured in the same assay at the same time point after administration; and/or 2020217894

(xx) the method achieves a greater GCase bioavailability in the liver, spleen, and/or bone marrow of the subject over a period of at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, or at least 35 weeks after administration when compared to the bioavailability measured in a subject administered an effective dose of a GCase enzyme replacement therapy, when measured in the same assay at the same time point after administration.

14. The method of claim 12 or claim 13, wherein: (a) the disease is Gaucher disease; or (b) the disease is Gaucher disease type I, II or III.

15. A method of reducing hexosylceramide and/or hexosylsphingosine levels in a subject suffering from a disease or condition associated with GCase deficiency, (a) the method comprising administering to the subject an effective amount of the polynucleotide of any one of claims 1 to 6, the viral particle of claim 7 or claim 8 or the composition of claim 9; or (b) the method comprising administering to the subject an effective amount of the polynucleotide of any one of claims 1 to 6, the viral particle of claim 7 or claim 8 or the composition of claim 9, wherein reducing hexosylceramide and/or hexosylsphingosine levels leads to the treatment of the disease or condition associated with GCase deficiency.

16. The method of claim 15, wherein: (i) the hexosylceramide and/or hexosylsphingosine levels are reduced by 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 2 to 3 times, 2 to 4 times, 2 to 5 times, 2 to 6 times, or 3 to 5 times when compared to the hexosylceramide and/or hexosylsphingosine levels at the time of administration of the polynucleotide of any one of claims 1 to 6, the viral particle of claim 7 or claim 8 or the 24 Jul 2025 composition of claim 9; (ii) (a) the reduction in hexosylceramide and/or hexosylsphingosine levels is greater than the reduction achieved in a subject administered an effective dose of a GCase enzyme replacement therapy, or (b) the reduction in hexosylceramide and/or hexosylsphingosine levels is greater than the reduction achieved in a subject administered an effective dose of a GCase enzyme 2020217894 replacement therapy when the hexosylceramide and/or hexosylsphingosine levels are measured at least 6 weeks, at least 8 weeks, at least 10 weeks or at least 12 weeks after administration; (iii) the hexosylceramide and/or hexosylsphingosine levels are measured in the macrophages of the subject; (iv) the hexosylceramide and/or hexosylsphingosine levels are measured in the spleen of the subject; (v) the hexosylceramide and/or hexosylsphingosine levels are measured in the liver of the subject; (vi) the hexosylceramide and/or hexosylsphingosine levels are measured in the serum of the subject; (vii) the hexosylceramide and/or hexosylsphingosine levels are measured by mass spectrometry; and/or (viii) the disease is Gaucher disease, or Gaucher disease type I, II or III.

17. The method of any one of claims 9 to 16, wherein the patient has antibodies or inhibitors to a recombinant GCase with which the patient has previously been treated as part of an enzyme replacement therapy.

18. Use of an effective amount of the polynucleotide of any one of claims 1 to 6, the viral particle of claim 7 or claim 8 or the composition of claim 9 in the manufacture of a medicament for treatment of: (i) Gaucher disease; (ii) Gaucher disease wherein the Gaucher disease is type I, II or III; and/or

(iii) Gaucher disease wherein the patient has antibodies or inhibitors to a recombinant 24 Jul 2025

GCase with which the patient has previously been treated as part of an enzyme replacement therapy.

19. Use of an effective amount of the polynucleotide of any one of claims 1 to 6, the viral particle of claim 7 or claim 8 or the composition of claim 9 in the manufacture of a medicament for reducing hexosylceramide and/or hexosylsphingosine levels in a 2020217894

subject suffering from a disease or condition associated with GCase deficiency, wherein reducing hexosylceramide and/or hexosylsphingosine levels leads to the treatment of the disease or condition associated with GCase deficiency.

WO wo 2020/161483 PCT/GB2020/050251 1/22

FIGURE 1

A LSP- LSP- FLF-PL01 S $ GBAwt GBAwt

B LSP- LSP- FLF-PL28 FLF-PL28 S $ GBAco

C LSP-L LSP-L GBAco FLF-PL64 FLF-PL64

FIGURE 2

A Naive Naive 6x1011 6x10¹¹ vg/kg

2x1012 vg/kg 2x1012 2x10¹² vg/kg

B 10000 (nmol/h/ml) activity GCase Serum I 1000 6x1012 vg/kg HI

I 2x1012 2x10¹² vg/kg

100 6x1011vg/kg 6x10 vg/kg

TO Naive K 10

4 8 12 weeks post AAV infusion

FIGURE 3 PL01 to relative change fold expression GCase 2.0

T 1.5 7 T

1.0

0.5 T

H

01 19 20 18 19 17 18 16 17 01 16 20 21 21 22 22 23 23 24 24 25 25 26 26 27 27 28 28 29 29 30 30 31 31 32 32 33 34 33 35 35 36 34 36 Construct (PL#)

WO wo 2020/161483 PCT/GB2020/050251 4/22

FIGURE 4

A 10000 (nmol/h/ml) activity GCase Serum 1000 6-fold

T

100

10

1 Naive 01 21 28 30 36 37

PL

B 10000 * * * FLF-PL28 (nmol/h/ml) activity GCase I 5-fold 5-fold 1000 FLF-PL01 (GBAwt)

100

Naive 10

1 0 4 8 12 36 Time (weeks post AAV infusion)

WO 2020/161483 2020/161483 PCT/GB2020/050251 5/22

FIGURE FIGURE 55

Naive Naive FLF-PL01 FLF-PL01 FLF-PL28 FLF-PL28

Spiger Spleen

Bone marrow

Bone

FIGURE 6

F4/80 (Macrophages) DAPI (Nuclear Counterstain) GBA

WO WO 2020/161483 2020/161483 PCT/GB2020/050251 PCT/GB2020/050251 7/22

FIGURE 7

P= 0.0001 10000 (nmol/h/ml) activity GCase Serum 1000 Service

100 Cases

10 Same

1 Naive PL28 PL64

WO wo 2020/161483 PCT/GB2020/050251 8/22

FIGURE 8

Spleen Bone Bone Lung

Naive

PL28

PL64

FIGURE FIGURE 99

SEQ ID NO: 1 - Codon-optimised Codon-optimised GBA GBA nucleotide nucleotide sequence sequence from from FLF-PL28, FLF-PL28, without without signal signal peptide portion

GCCAGGCCCTGCATCCCTAAGAGCTTTGGCTACAGCTCTGTGGTGTGTGTGTGCAATGCCACCTAC GTGACAGCTTTGACCCCCCCACCTTTCCTGCCCTGGGCACCTTCAGCAGATATGAGAGCACCAGG TGTGACAGCTTTGACCCCCCCACCTTTCCTGCCCTGGGCACCTTCAGCAGATATGAGAGCACCAGGT CTGGGAGGAGGATGGAGCTGAGCATGGGGCCCATCCAGGCTAATCACACTGGCACTGGCCTGCTG CTGGGAGGAGGATGGAGCTGAGCATGGGGCCCATCCAGGCTAATCACACTGGCACTGGCCTGCTO CTGACCCTGCAGCCTGAGCAGAAGTTCCAGAAAGTAAAGGGCTTTGGAGGGGCCATGACTGATGO CTGACCCTGCAGCCTGAGCAGAAGTTCCAGAAAGTAAAGGGCTTTGGAGGGGCCATGACTGATGCT GCTGCTCTGAACATCCTGGCCCTGAGCCCCCCTGCCCAGAATCTGCTGCTGAAGAGCTACTTCTC GCTGCTCTGAACATCCTGGCCCTGAGCCCCCCTGCCCAGAATCTGCTGCTGAAGAGCTACTTCTCTG AGGAGGGCATTGGCTATAACATCATCAGGGTGCCCATGGCCAGCTGTGACTTCAGCATCAGGACCT AGGAGGGCATTGGCTATAACATCATCAGGGTGCCCATGGCCAGCTGTGACTTCAGCATCAGGACCT CACCTATGCTGACACCCCTGATGATTTCCAGCTGCACAACTTCAGCCTGCCTGAGGAGGATACCAA ACACCTATGCTGACACCCCTGATGATTTCCAGCTGCACAACTTCAGCCTGCCTGAGGAGGATACCAA GCTGAAGATCCCACTGATCCACAGGGCTCTGCAGCTGGCCCAGAGGCCTGTGAGCCTGCTGGCCAG GCTGAAGATCCCACTGATCCACAGGGCTCTGCAGCTGGCCCAGAGGCCTGTGAGCCTGCTGGCCAG CCCCTGGACCAGCCCCACTTGGCTGAAGACCAATGGGGCTGTGAATGGGAAGGGGAGCCTGAAGG CCCCTGGACCAGCCCCACTTGGCTGAAGACCAATGGGGCTGTGAATGGGAAGGGGAGCCTGAAGG GACAGCCTGGAGACATCTACCACCAGACCTGGGCCAGATACTTTGTGAAGTTCCTGGATGCCTATGC GACAGCCTGGAGACATCTACCACCAGACCTGGGCCAGATACTTTGTGAAGTTCCTGGATGCCTATGO TGAGCACAAGCTGCAGTTCTGGGCTGTGACTGCTGAGAATGAGCCTTCTGCTGGGCTGCTGTCTGG TGAGCACAAGCTGCAGTTCTGGGCTGTGACTGCTGAGAATGAGCCTTCTGCTGGGCTGCTGTCTGG TACCCCTTCCAATGCCTGGGCTTCACCCCTGAGCATCAGAGGGACTTCATTGCCAGGGACCTGGO CTACCCCTTCCAATGCCTGGGCTTCACCCCTGAGCATCAGAGGGACTTCATTGCCAGGGACCTGGG CCCTACCCTGGCCAACAGCACTCACCATAATGTTAGGCTGCTGATGCTGGATGACCAGAGGCTGCT CCCTACCCTGGCCAACAGCACTCACCATAATGTTAGGCTGCTGATGCTGGATGACCAGAGGCTGCT GCTGCCCCACTGGGCTAAGGTGGTGCTGACTGACCCTGAGGCTGCTAAATATGTGCATGGCATTG GCTGCCCCACTGGGCTAAGGTGGTGCTGACTGACCCTGAGGCTGCTAAATATGTGCATGGCATTGC GTGCATTGGTACCTGGACTTTCTGGCTCCTGCCAAGGCCACCCTGGGGGAGACCCACAGGCTGT TGTGCATTGGTACCTGGACTTTCTGGCTCCTGCCAAGGCCACCCTGGGGGAGACCCACAGGCTGTT CCCCAACACCATGCTGTTTGCCTCTGAGGCCTGTGTGGGCAGCAAGTTCTGGGAGCAGTCTGTGAG CCCCAACACCATGCTGTTTGCCTCTGAGGCCTGTGTGGGCAGCAAGTTCTGGGAGCAGTCTGTGAG GCTGGGCAGCTGGGATAGGGGGATGCAGTACAGCCACAGCATCATCACCAACCTGCTGTACCATGT GCTGGGCAGCTGGGATAGGGGGATGCAGTACAGCCACAGCATCATCACCAACCTGCTGTACCATGT GGTGGGCTGGACTGACTGGAACCTGGCCCTGAACCCTGAGGGAGGACCTAACTGGGTCAGAAACT GGTGGGCTGGACTGACTGGAACCTGGCCCTGAACCCTGAGGGAGGACCTAACTGGGTCAGAAACT TGTGGACAGCCCCATCATTGTGGACATCACCAAGGACACCTTTTACAAGCAGCCCATGTTCTACCAC TGTGGACAGCCCCATCATTGTGGACATCACCAAGGACACCTTTTACAAGCAGCCCATGTTCTACCAC CTGGGCCACTTCAGCAAGTTCATCCCTGAGGGCAGCCAGAGAGTGGGGCTGGTGGCCAGCCAGAA AATGACCTGGATGCTGTGGCTCTGATGCATCCTGATGGCTCTGCTGTGGTGGTGGTGCTGAACA GAATGACCTGGATGCTGTGGCTCTGATGCATCCTGATGGCTCTGCTGTGGTGGTGGTGCTGAACAG GAGCTCTAAGGATGTGCCTCTGACCATCAAGGATCCTGCTGTGGGCTTCCTGGAGACCATCAGCCC TGGCTACAGCATCCACACCTACCTGTGGAGGAGGCAGTGA TGGCTACAGCATCCACACCTACCTGTGGAGGAGGCAGTGA SEQ ID NO: 2 - Codon-optimised Codon-optimised GBA GBA nucleotide nucleotide sequence sequence from from FLF-PL21, FLF-PL21, without without signal signal peptide portion

GCCAGGCCCTGTATCCCTAAGAGCTTtGGCTACAGCTCAGTaGTtTGTGTCTGTAATGCCACATACT GCCAGGCCCTGTATCCCTAAGAGCTTEGGCTACAGCTCAGTaGTETGTGTCTGTAATGCCACATACTO TGACTCCTTtGACCCCCCTACCTTCCCTGCCCTGGGAACCTTCAGCAGaTATGAGTCAACAAGaTCA TGACTCCTTtGACCCCCCTACCTTCCCTGCCCTGGGAACCTTCAGCAGaTATGAGTCAACAAGaTCAG GAAGGAGGATGGAGCTGTCAATGGGACCCATCCAGGCTAATCACACAGGCACAGGCCTGCTGCTC GAAGGAGGATGGAGCTGTCAATGGGACCCATCCAGGCTAATCACACAGGCACAGGCCTGCTGCTGA CCCTGCAGCCAGAACAGAAGTTCCAGAAaGTGAAGGGATTtGGAGGAGCCATGACAGATGCTGCTG CTCTCAACATCCTGGCCCTGTCACCCCCTGCCCAGAATCTGCTGCTGAAGTCATACTTCTCTGAAG CTCTCAACATCCTGGCCCTGTCACCCCCTGCCCAGAATCTGCTGCTGAAGTCATACTTCTCTGAAGA AGGAATtGGATATAACATCATCAGGGTGCCCATGGCCAGCTGTGACTTCTCCATCAGGACCTACAC AGGAATtGGATATAACATCATCAGGGTGCCCATGGCCAGCTGTGACTTCTCCATCAGGACCTACACO TATGCtGACACCCCTGATGATTTCCAGCTGCACAACTTCAGCCTCCCAGAGGAAGATACCAAGCTCAA TATGCtGACACCCCTGATGATTTCCAGCTGCACAACTTCAGCCTCCCAGAGGAAGATACCAAGCTCAA GATCCCtCTGATaCAtAGgGCaCTGCAGCTGGCCCAGAGGCCtGTGTCACTCCTGGCCAGCCCCTGGA GATCCCtCTGATaCAtAGgGCaCTGCAGCTGGCCCAGAGGCCtGTGTCACTCCTGGCCAGCCCCTGGA CATCACCCACTTGGCTCAAGACCAATGGAGCtGTGAATGGAAAGGGATCACTCAAGGGACAGCCtGO CATCACCCACTTGGCTCAAGACCAATGGAGCtGTGAATGGAAAGGGATCACTCAAGGGACAGCCtGG AGACATCTACCACCAGACCTGGGCCAGaTACTTtGTGAAGTTCCTGGATGCCTATGCTGAGCACAA AGACATCTACCACCAGACCTGGGCCAGaTACTTEGTGAAGTTCCTGGATGCCTATGCTGAGCACAAG TGCAGTTCTGGGCaGTGACAGCTGAAAATGAGCCTTCTGCTGGACTGCTGTCAGGATACCCCTTO CTGCAGTTCTGGGCaGTGACAGCTGAAAATGAGCCTTCTGCTGGACTGCTGTCAGGATACCCCTTCC AGTGTCTGGGCTTCACCCCTGAACATCAGAGGGACTTCATtGCCAGGGACCTGGGACCTACCCTtG AGTGTCTGGGCTTCACCCCTGAACATCAGAGGGACTTCATEGCCAGGGACCTGGGACCTACCCTEGC AACTCAACTCACCACAATGTCAGGCTGCTCATGCTGGATGACCAGAGGCTGCTGCTGCCCCACTGG CAACTCAACTCACCACAATGTCAGGCTGCTCATGCTGGATGACCAGAGGCTGCTGCTGCCCCACTGG GCCAAGGTGGTGCTGACAGACCCAGAAGCtGCTAAaTATGTGCATGGCATtGCTGTGCATTGGTACC GCCAAGGTGGTGCTGACAGACCCAGAAGCtGCTAAaTATGTGCATGGCATtGCTGTGCATTGGTACC GGACTTCCTGGCTCCAGCCAAGGCCACCCTGGGAGAGACACACAGGCTGTTCCCCAACACCATGC TGGACTTCCTGGCTCCAGCCAAGGCCACCCTGGGAGAGACACACAGGCTGTTCCCCAACACCATGCT TtGCCTCtGAGGCCTGTGTGGGCTCCAAGTTCTGGGAGCAGTCAGTGAGGCTGGGCTCCTGGGA CTTLGCCTCtGAGGCCTGTGTGGGCTCCAAGTTCTGGGAGCAGTCAGTGAGGCTGGGCTCCTGGGA AGGGGAATGCAGTACAGCCACAGCATCATCACAAACCTCCTGTACCATGTGGTgGGCTGGACtG/ TAGGGGAATGCAGTACAGCCACAGCATCATCACAAACCTCCTGTACCATGTGGTgGGCTGGACtGAC TGGAACCTGGCCCTGAACCCtGAAGGAGGACCCAAcTGGGTcagaAAtTTtGTgGACTCACCCATCAT TGGAACCTGGCCCTGAACCCtGAAGGAGGACCCAAcTGGGTcagaAAtTTtGTgGACTCACCCATCATt GTGGACATCACCAAGGACACATTCTACAAGCAGCCCATGTTCTACCACCTGGGCCACTTCAGCAAG] GTGGACATCACCAAGGACACATTCTACAAGCAGCCCATGTTCTACCACCTGGGCCACTTCAGCAAGT TCATCCCTGAGGGCTCCCAGAGGGTGGGACTGGTGGCCTCACAGAAGAAtGACCTGGAtGCaGTGG TCATCCCTGAGGGCTCCCAGAGGGTGGGACTGGTGGCCTCACAGAAGAAtGACCTGGAtGCaGTGG

CCTGATGCATCCtGATGGCTCTGCTGTGGTGGTtGTGCTGAAtAGaTCCTCTAAGGATGTGCCTCT CCCTGATGCATCCtGATGGCTCTGCTGTGGTGGTtGTGCTGAAtAGaTCCTCTAAGGATGTGCCTCT GACCATCAAGGATCCTGCTGTGGGCTTCCTGGAGACAATCTCACCTGGCTACTCCATCCACACCTAC CTGTGGAGGAGGCAGTGA SEQ ID NO: 3 - Codon-optimised Codon-optimised GBA GBA nucleotide nucleotide sequence sequence from from FLF-PL30, FLF-PL30, without without signal signal peptide portion

AGGCCCTGCATCCCTAAGAGCTTTGGCTACAGCTCTGTGGTGTGTGTGTGCAATGCCACATA GCCAGGCCCTGCATCCCTAAGAGCTTTGGCTACAGCTCTGTGGTGTGTGTGTGCAATGCCACATAC TGTGACTCCTTTGACCCCCCCACCTTTCCTGCCCTGGGCACaTTctccAGaTATGAGAGCACAAGAT TGTGACTCCTTTGACCCCCCCACCTTTCCTGCCCTGGGCACaTTctccAGaTATGAGAGCACAAGATC TGGGAGAAGGATGGAGCTGAGCATGGGGCCCATCCAGGCTAATCACACTGGCACAGGCCTGCTGCT TGGGAGAAGGATGGAGCTGAGCATGGGGCCCATCCAGGCTAATCACACTGGCACAGGCCTGCTGCT GACCCTGCAGCCTGAACAGAAGTTTCAGAAaGTGAAGGGATTTGGAGGGGCCATGACAGATGCTGC GACCCTGCAGCCTGAACAGAAGTTTCAGAAaGTGAAGGGATTTGGAGGGGCCATGACAGATGCTGC TGCTCTGAATATCCTGGCCCTGTCACCCCCTGCCCAGAATCTGCTGCTGAAGAGCTACTTTTCAGA TGCTCTGAATATCCTGGCCCTGTCACCCCCTGCCCAGAATCTGCTGCTGAAGAGCTACTTTTCAGAA GAAGGAATTGGATATAATATCATCAGAGTGCCCATGGCCAGCTGTGACTTTTCCATCAGAACCTACA GAAGGAATTGGATATAATATCATCAGAGTGCCCATGGCCAGCTGTGACTTTTCCATCAGAACCTACA CCTATGCAGACACCCCTGATGATILTCAGCTGCACAATTTTAGCCTGCCTGAGGAAGATACCAAGO CCTATGCAGACACCCCTGATGATTTTCAGCTGCACAATTTTAGCCTGCCTGAGGAAGATACCAAGCT AAGATACCCCTGATTCACAGGGCCCTGCAGCTGGCCCAGAGGCCTGTTTCACTGCTGGCCAGCCO GAAGATACCCCTGATTCACAGGGCCCTGCAGCTGGCCCAGAGGCCTGTTTCACTGCTGGCCAGCCC CTGGACATCACCCACCTGGCTGAAGACCAATGGAGCTGTGAATGGGAAGGGGTCACTGAAGGGACA CTGGAGACATCTACCACCAGACCTGGGCCAGATACTTTGTGAAGTTTCTGGATGCCTATGC GCCTGGAGACATCTACCACCAGACCTGGGCCAGATACTTTGTGAAGTTTCTGGATGCCTATGCTGA GCACAAGCTGCAGTTTTGGGCAGTGACAGCTGAAAATGAGCCTTCAGCTGGGCTGCTGTCAGGAT GCACAAGCTGCAGTTTTGGGCAGTGACAGCTGAAAATGAGCCTTCAGCTGGGCTGCTGTCAGGATA CCCTTTCAGTGCCTGGGCTTTACCCCTGAACATCAGAGGGACTTTATTGCCAGGGACCTGGGCC CCCCTTTCAGTGCCTGGGCTTTACCCCTGAACATCAGAGGGACTTTATTGCCAGGGACCTGGGCCCT ACCCTGGCCAATAGCACCCACCAtAATGTgAGgttgCTGATGCTGGATGACCAGAGGCTGCTGCTGCC ACCCTGGCCAATAGCACCCACCAtAATGTgAGgttgCTGATGCTGGATGACCAGAGGCTGCTGCTGCC CCACTGGGCAAAGGTGGTGCTGACAGACCCTGAAGCAGCTAAaTATGTTCATGGCATTGCTGTGCA CCACTGGGCAAAGGTGGTGCTGACAGACCCTGAAGCAGCTAAaTATGTTCATGGCATTGCTGTGCA TGGTACCTGGACTTTCTGGCTCCTGCCAAGGCCACCCTGGGGGAGACACACAGGCTGTTTCCCA TTGGTACCTGGACTTTCTGGCTCCTGCCAAGGCCACCCTGGGGGAGACACACAGGCTGTTTCCCAA TACCATGCTGTTTGCCTCtGAGGCCTGTGTGGGCTCCAAGTTTTGGGAGCAGTCTGTGAGGCTGGG TACCATGCTGTTTGCCTCtGAGGCCTGTGTGGGCTCCAAGTTTTGGGAGCAGTCTGTGAGGCTGGG TCCTGGGATAGAGGGATGCAGTACAGCCACAGCATCATCACCAATCTGCTGTACCATGTGGTGGG CTCCTGGGATAGAGGGATGCAGTACAGCCACAGCATCATCACCAATCTGCTGTACCATGTGGTGGG CTGGACTGACTGGAATCTGGCCCTGAATCCTGAAGGAGGACCtAACTGGGTcAGgAATTTTGTGGAC AGCCCCATCATTGTGGACATCACCAAGGACACCTTTTACAAGCAGCCCATGTTTTACCACCTGGGCC AGCCCCATCATTGTGGACATCACCAAGGACACCTTTTACAAGCAGCCCATGTTTTACCACCTGGGCO ACTTTAGCAAGTTTATTCCTGAGGGCTCCCAGAGAGTGGGGCTGGTTGCCAGCCAGAAGAATGAC ACTTTAGCAAGTTTATTCCTGAGGGCTCCCAGAGAGTGGGGCTGGTTGCCAGCCAGAAGAATGAC TGGATGCAGTGGCACTGATGCATCCTGATGGCTCAGCTGTTGTGGTGGTGCTGAATAGATCCAGCA TGGATGCAGTGGCACTGATGCATCCTGATGGCTCAGCTGTTGTGGTGGTGCTGAATAGATCCAGCA AGGATGTGCCTCTGACCATCAAGGATCCTGCTGTGGGCTTTCTGGAGACAATCTCACCTGGCTACT AGGATGTGCCTCTGACCATCAAGGATCCTGCTGTGGGCTTTCTGGAGACAATCTCACCTGGCTACTO CATTCACACCTACCTGTGGAGAAGGCAGTGA CATTCACACCTACCTGTGGAGAAGGCAGTGA SEQ ID NO: 4 - Codon-optimised Codon-optimised GBA GBA nucleotide nucleotide sequence sequence from from FLF-PL36, FLF-PL36, without without signal signal peptide portion

GCCAGGCCTTGCATCCCAAAGTCTTTCGGCTACAGCTCCGTGGTGTGCGTGTGCAACGCCACCTATT TGACTCCTTCGATCCCCCTACCTTTCCCGCCCTGGGCACATTTTCTAGATACGAGTCTACACGCA GTGACTCCTTCGATCCCCCTACCTTTCCCGCCCTGGGCACATTTTCTAGATACGAGTCTACACGGAG EGGCCGGAGAATGGAGCTGAGCATGGGCCCTATCCAGGCCAATCACACAGGAACAGGCCTGCTGG CGGCCGGAGAATGGAGCTGAGCATGGGCCCTATCCAGGCCAATCACACAGGAACAGGCCTGCTGCT GACCCTGCAGCCAGAGCAGAAGTTCCAGAAGGTGAAGGGCTTTGGCGGAGCCATGACAGATGCA GACCCTGCAGCCAGAGCAGAAGTTCCAGAAGGTGAAGGGCTTTGGCGGAGCCATGACAGATGCAGG CGCCCTGAACATCCTGGCCCTGTCCCCACCCGCCCAGAATCTGCTGCTGAAGTCCTACTTCTCTGAG GAGGGCATCGGCTATAACATCATCCGGGTGCCCATGGCCAGCTGCGACTTTTCCATCAGAACCTA GAGGGCATCGGCTATAACATCATCCGGGTGCCCATGGCCAGCTGCGACTTTTCCATCAGAACCTACA CATATGCCGATACCCCTGACGATTTCCAGCTGCACAATTITTCCCTGCCAGAGGAGGATACAAAGO CATATGCCGATACCCCTGACGATTTCCAGCTGCACAATTTTTCCCTGCCAGAGGAGGATACAAAGCT GAAGATCCCCCTGATTCACCGGGCCCTGCAGCTGGCACAGCGGCCCGTGAGCCTGCTGGCCAGCC GAAGATCCCCCTGATTCACCGGGCCCTGCAGCTGGCACAGCGGCCCGTGAGCCTGCTGGCCAGCCC CTGGACCTCCCCTACATGGCTGAAGACCAACGGCGCCGTGAATGGCAAGGGCTCTCTGAAGGGACA CTGGACCTCCCCTACATGGCTGAAGACCAACGGCGCCGTGAATGGCAAGGGCTCTCTGAAGGGACA GCCTGGCGACATCTACCACCAGACATGGGCCAGATATTTCGTGAAGTTTCTGGATGCCTACGCCGA GCCTGGCGACATCTACCACCAGACATGGGCCAGATATTTCGTGAAGTTTCTGGATGCCTACGCCGA GCACAAGCTGCAGTTCTGGGCCGTGACAGCAGAGAATGAGCCTTCTGCCGGCCTGCTGAGCGGCTA GCACAAGCTGCAGTTCTGGGCCGTGACAGCAGAGAATGAGCCTTCTGCCGGCCTGCTGAGCGGCTA CCCTTCCAGTGCCTGGGCTTTACACCTGAGCACCAGCGGGACTTTATCGCCAGAGATCTGGGCCC TCCCTTCCAGTGCCTGGGCTTTACACCTGAGCACCAGCGGGACTTTATCGCCAGAGATCTGGGCCC AACCCTGGCCAACTCCACACACCACAATGTGAGGCTGCTGATGCTGGACGATCAGCGCCTGCTGCT AACCCTGGCCAACTCCACACACCACAATGTGAGGCTGCTGATGCTGGACGATCAGCGCCTGCTGCT GCCTCACTGGGCCAAGGTGGTGCTGACCGACCCAGAGGCCGCCAAGTACGTGCACGGCATCGCCGT GCCTCACTGGGCCAAGGTGGTGCTGACCGACCCAGAGGCCGCCAAGTACGTGCACGGCATCGCCGT GCACTGGTATCTGGATTTCCTGGCACCTGCAAAGGCCACCCTGGGAGAGACACACCGGCTGTTCCC GCACTGGTATCTGGATTTCCTGGCACCTGCAAAGGCCACCCTGGGAGAGACACACCGGCTGTTCCC TAACACCATGCTGTTTGCCAGCGAGGCCTGCGTGGGCTCCAAGTTTTGGGAGCAGTCCGTGAGGCT TAACACCATGCTGTTTGCCAGCGAGGCCTGCGTGGGCTCCAAGTTTTGGGAGCAGTCCGTGAGGCT GGGATCTTGGGACAGAGGCATGCAGTACTCCCACTCTATCATCACCAATCTGCTGTATCACGTGGT GGCTGGACAGACTGGAACCTGGCCCTGAATCCAGAGGGCGGCCCCAACTGGGTGAGAAATTTCGTC GGCTGGACAGACTGGAACCTGGCCCTGAATCCAGAGGGCGGCCCCAACTGGGTGAGAAATTTCGTG GATAGCCCCATCATCGTGGACATCACCAAGGATACATTCTACAAGCAGCCAATGTTTTATCACCTG GATAGCCCCATCATCGTGGACATCACCAAGGATACATTCTACAAGCAGCCAATGTTTTATCACCTGG CCACTTCTCTAAGTTTATCCCTGAGGGCAGCCAGAGGGTGGGCCTGGTGGCCAGCCAGAAGAAG GCCACTTCTCTAAGTTTATCCCTGAGGGCAGCCAGAGGGTGGGCCTGGTGGCCAGCCAGAAGAACG

ACCTGGATGCCGTGGCCCTGATGCACCCTGATGGCTCCGCCGTGGTGGTGGTGCTGAATCGCTCT/ ACCTGGATGCCGTGGCCCTGATGCACCCTGATGGCTCCGCCGTGGTGGTGGTGCTGAATCGCTCTA GCAAGGACGTGCCTCTGACCATCAAGGATCCAGCCGTGGGATTTCTGGAGACTATTTCACCTGGCT GCAAGGACGTGCCTCTGACCATCAAGGATCCAGCCGTGGGATTTCTGGAGACTATTTCACCTGGCT ATTCAATTCATACCTACCTGTGGAGGAGGCAGTGA ATTCAATTCATACCTACCTGTGGAGGAGGCAGTGA SEQ ID NO: 5 - Codon-optimised Codon-optimised GBA GBA nucleotide nucleotide sequence sequence from from FLF-PL28, FLF-PL28, with with signal signal peptide portion

ATGGAGTTLTCAAGTCCTTCCAGAGAGGAATGTCCCAAGCCTTTGAGTAGGGTAAGCATCATGGO ATGGAGTTTTCAAGTCCTTCCAGAGAGGAATGTCCCAAGCCTTTGAGTAGGGTAAGCATCATGGCT GCAGCCTCACAGGATTGCTTCTACTTCAGGCAGTGTCGTGGGCATCAGGTGCCAGGCCCTGCAT GGCAGCCTCACAGGATTGCTTCTACTTCAGGCAGTGTCGTGGGCATCAGGTGCCAGGCCCTGCATC CCTAAGAGCTTTGGCTACAGCTCTGTGGTGTGTGTGTGCAATGCCACCTACTGTGACAGCTTTGA CCTAAGAGCTTTGGCTACAGCTCTGTGGTGTGTGTGTGCAATGCCACCTACTGTGACAGCTTTGAC CCCCCACCTTTCCTGCCCTGGGCACCTTCAGCAGaTATGAGAGCACCAGGTCTGGGAGGAGGATO CCCCCCACCTTTCCTGCCCTGGGCACCTTCAGCAGaTATGAGAGCACCAGGTCTGGGAGGAGGATG GAGCTGAGCATGGGGCCCATCCAGGCTAATCACACTGGCACTGGCCTGCTGCTGACCCTGCAGCCT GAGCTGAGCATGGGGCCCATCCAGGCTAATCACACTGGCACTGGCCTGCTGCTGACCCTGCAGCCT GAGCAGAAGTTCCAGAAaGTaAAGGGCTTTGGAGGGGCCATGACTGATGCTGCTGCTCTGAACAT GAGCAGAAGTTCCAGAAaGTaAAGGGCTTTGGAGGGGCCATGACTGATGCTGCTGCTCTGAACATO :TGGCCCTGAGCCCCCCTGCCCAGAATCTGCTGCTGAAGAGCTACTTCTCTGAGGAGGGCATTGO CTGGCCCTGAGCCCCCCTGCCCAGAATCTGCTGCTGAAGAGCTACTTCTCTGAGGAGGGCATTGGG TATAACATCATCAGGGTGCCCATGGCCAGCTGTGACTTCAGCATCAGGACCTACACCTATGCTGAC TATAACATCATCAGGGTGCCCATGGCCAGCTGTGACTTCAGCATCAGGACCTACACCTATGCTGACA CCCCTGATGATTTCCAGCTGCACAACTTCAGCCTGCCTGAGGAGGATACCAAGCTGAAGATCCCaCT CCCCTGATGATTTCCAGCTGCACAACTTCAGCCTGCCTGAGGAGGATACCAAGCTGAAGATCCCaCT GATCCACAGGGCtCTGCAGCTGGCCCAGAGGCCTGTGAGCCTGCTGGCCAGCCCCTGGACCAGCO GATCCACAGGGCtCTGCAGCTGGCCCAGAGGCCTGTGAGCCTGCTGGCCAGCCCCTGGACCAGCCC CACTTGGCTGAAGACCAATGGGGCTGTGAATGGGAAGGGGAGCCTGAAGGGACAGCCTGGAGACA TCTACCACCAGACCTGGGCCAGATACTTTGTGAAGTTCCTGGATGCCTATGCTGAGCACAAGCTG TCTACCACCAGACCTGGGCCAGATACTTTGTGAAGTTCCTGGATGCCTATGCTGAGCACAAGCTGCA GTTCTGGGCTGTGACTGCTGAGAATGAGCCTTCTGCTGGGCTGCTGTCTGGCTACCCCTTCCAaT GTTCTGGGCTGTGACTGCTGAGAATGAGCCTTCTGCTGGGCTGCTGTCTGGCTACCCCTTCCATG CCTGGGCTTCACCCCTGAGCATCAGAGGGACTTCATTGCCAGGGACCTGGGCCCTACCCTGGCCAA CCTGGGCTTCACCCCTGAGCATCAGAGGGACTTCATTGCCAGGGACCTGGGCCCTACCCTGGCCAA CAGCACTCACCAtAATGTtAGGCTGCTGATGCTGGATGACCAGAGGCTGCTGCTGCCCCACTGGGCT CAGCACTCACCAtAATGTtAGGCTGCTGATGCTGGATGACCAGAGGCTGCTGCTGCCCCACTGGGCT AAGGTGGTGCTGACTGACCCTGAGGCTGCTAAaTATGTGCATGGCATTGCTGTGCATTGGTACCT AAGGTGGTGCTGACTGACCCTGAGGCTGCTAAaTATGTGCATGGCATTGCTGTGCATTGGTACCTG BACTTTCTGGCTCCTGCCAAGGCCACCCTGGGGGAGACCCACAGGCTGTTCCCCAACACCATGCTG GACTTTCTGGCTCCTGCCAAGGCCACCCTGGGGGAGACCCACAGGCTGTTCCCCAACACCATGCTG TTGCCTCTGAGGCCTGTGTGGGCAGCAAGTTCTGGGAGCAGTCTGTGAGGCTGGGCAGCTGGGA ITTGCCTCTGAGGCCTGTGTGGGCAGCAAGTTCTGGGAGCAGTCTGTGAGGCTGGGCAGCTGGGA TAGGGGGATGCAGTACAGCCACAGCATCATCACCAACCTGCTGTACCATGTGGTGGGCTGGACTGA TAGGGGGATGCAGTACAGCCACAGCATCATCACCAACCTGCTGTACCATGTGGTGGGCTGGACTGA CTGGAACCTGGCCCTGAACCCTGAGGGAGGACCtAAcTGGGTcAGaAACTTTGTGGACAGCCCCAT CTGGAACCTGGCCCTGAACCCTGAGGGAGGACCtAAcTGGGTcAGaAACTTTGTGGACAGCCCCATC ATTGTGGACATCACCAAGGACACCTTTTACAAGCAGCCCATGTTCTACCACCTGGGCCACTTCAGO ATTGTGGACATCACCAAGGACACCTTTTACAAGCAGCCCATGTTCTACCACCTGGGCCACTTCAGCA AGTTCATCCCTGAGGGCAGCCAGAGAGTGGGGCTGGTGGCCAGCCAGAAGAATGACCTGGATGCT IGGCTCTGATGCATCCTGATGGCTCTGCTGTGGTGGTGGTGCTGAACAGGAGCTCTAAGGATGT GTGGCTCTGATGCATCCTGATGGCTCTGCTGTGGTGGTGGTGCTGAACAGGAGCTCTAAGGATGTG CCTCTGACCATCAAGGATCCTGCTGTGGGCTTCCTGGAGACCATCAGCCCTGGCTACAGCATCCACA CCTACCTGTGGAGGAGGCAGTGA CCTACCTGTGGAGGAGGCAGTGA SEQ ID NO: 6 - Codon-optimised Codon-optimised GBA GBA nucleotide nucleotide sequence sequence from from FLF-PL21, FLF-PL21, with with signal signal peptide portion

GGAGTTTTCAAGTCCTTCCAGAGAGGAATGTCCCAAGCCTTTGAGTAGGGTAAGCATCAT ATGGAGTTTTCAAGTCCTTCCAGAGAGGAATGTCCCAAGCCTTTGAGTAGGGTAAGCATCATGGCT GGCAGCCTCACAGGATTGCTTCTACTTCAGGCAGTGTCGTGGGCATCAGGTGCCAGGCCCTGTAT GGCAGCCTCACAGGATTGCTTCTACTTCAGGCAGTGTCGTGGGCATCAGGTGCCAGGCCCTGTATC CCTAAGAGCTTtGGCTACAGCTCAGTaGTtTGTGTCTGTAATGCCACATACTGTGACTCCTTtGACC CCTAAGAGCTTtGGCTACAGCTCAGTaGTtTGTGTCTGTAATGCCACATACTGTGACTCCTTEGACCO CCCTACCTTCCCTGCCCTGGGAACCTTCAGCAGaTATGAGTCAACAAGaTCAGGAAGGAGGATGG, CCCTACCTTCCCTGCCCTGGGAACCTTCAGCAGaTATGAGTCAACAAGaTCAGGAAGGAGGATGGAG CTGTCAATGGGACCCATCCAGGCTAATCACACAGGCACAGGCCTGCTGCTGACCCTGCAGCCAGAAG CTGTCAATGGGACCCATCCAGGCTAATCACACAGGCACAGGCCTGCTGCTGACCCTGCAGCCAGAAG AGAAGTTCCAGAAaGTGAAGGGATTtGGAGGAGCCATGACAGATGCTGCTGCTCTCAACATCCTGGC AGAAGTTCCAGAAaGTGAAGGGATTtGGAGGAGCCATGACAGATGCTGCTGCTCTCAACATCCTGGC CCTGTCACCCCCTGCCCAGAATCTGCTGCTGAAGTCATACTTCTCTGAAGAAGGAATtGGATATAAC CCTGTCACCCCCTGCCCAGAATCTGCTGCTGAAGTCATACTTCTCTGAAGAAGGAATtGGATATAAG ATCATCAGGGTGCCCATGGCCAGCTGTGACTTCTCCATCAGGACCTACACCTATGCtGACACCCCTG ATGATTTCCAGCTGCACAACTTCAGCCTCCCAGAGGAAGATACCAAGCTCAAGATCCCtCTGATaCAt ATGATTTCCAGCTGCACAACTTCAGCCTCCCAGAGGAAGATACCAAGCTCAAGATCCCECTGATaCAt AGgGCaCTGCAGCTGGCCCAGAGGCCtGTGTCACTCCTGGCCAGCCCCTGGACATCACCCACTTGGC AGgGCaCTGCAGCTGGCCCAGAGGCCtGTGTCACTCCTGGCCAGCCCCTGGACATCACCCACTTGGC CAAGACCAATGGAGCtGTGAATGGAAAGGGATCACTCAAGGGACAGCCtGGAGACATCTACCACO TCAAGACCAATGGAGCtGTGAATGGAAAGGGATCACTCAAGGGACAGCCtGGAGACATCTACCACCA GACCTGGGCCAGaTACTTtGTGAAGTTCCTGGATGCCTATGCTGAGCACAAGCTGCAGTTCTGGGCa GACCTGGGCCAGaTACTTtGTGAAGTTCCTGGATGCCTATGCTGAGCACAAGCTGCAGTTCTGGGCA GTGACAGCTGAAAATGAGCCTTCTGCTGGACTGCTGTCAGGATACCCCTTCCAGTGTCTGGGCTTC GTGACAGCTGAAAATGAGCCTTCTGCTGGACTGCTGTCAGGATACCCCTTCCAGTGTCTGGGCTTC ACCCCTGAACATCAGAGGGACTTCATtGCCAGGGACCTGGGACCTACCCTtGCCAACTCAACTCACCA ACCCCTGAACATCAGAGGGACTTCATEGCCAGGGACCTGGGACCTACCCTtGCCAACTCAACTCACCA CAATGTCAGGCTGCTCATGCTGGATGACCAGAGGCTGCTGCTGCCCCACTGGGCCAAGGTGGTGCT GACAGACCCAGAAGCtGCTAAaTATGTGCATGGCATtGCTGTGCATTGGTACCTGGACTTCCTGGCT GACAGACCCAGAAGCtGCTAAaTATGTGCATGGCATtGCTGTGCATTGGTACCTGGACTTCCTGGCT CCAGCCAAGGCCACCCTGGGAGAGACACACAGGCTGTTCCCCAACACCATGCTCTTtGCCTCtGAGO CCAGCCAAGGCCACCCTGGGAGAGACACACAGGCTGTTCCCCAACACCATGCTCTTEGCCTCGAGG CCTGTGTGGGCTCCAAGTTCTGGGAGCAGTCAGTGAGGCTGGGCTCCTGGGATAGGGGAATGCAG

CAGCCACAGCATCATCACAAACCTCCTGTACCATGTGGTgGGCTGGACtGACTGGAACCTGO TACAGCCACAGCATCATCACAAACCTCCTGTACCATGTGGTgGGCTGGACtGACTGGAACCTGGCCC TGAACCCtGAAGGAGGACCCAAcTGGGTcagaAAtTTtGTgGACTCACCCATCATtGTGGACATCACO AGGACACATTCTACAAGCAGCCCATGTTCTACCACCTGGGCCACTTCAGCAAGTTCATCCCTGAGGO AGGACACATTCTACAAGCAGCCCATGTTCTACCACCTGGGCCACTTCAGCAAGTTCATCCCTGAGGG TCCCAGAGGGTGGGACTGGTGGCCTCACAGAAGAAtGACCTGGAtGCaGTGGCCCTGATGCATCC CTCCCAGAGGGTGGGACTGGTGGCCTCACAGAAGAAtGACCTGGAtGCaGTGGCCCTGATGCATCCE GATGGCTCTGCTGTGGTGGTtGTGCTGAAtAGaTCCTCTAAGGATGTGCCTCTGACCATCAAGGATC GATGGCTCTGCTGTGGTGGTtGTGCTGAAtAGaTCCTCTAAGGATGTGCCTCTGACCATCAAGGATC CTGCTGTGGGCTTCCTGGAGACAATCTCACCTGGCTACTCCATCCACACCTACCTGTGGAGGAGGC CTGCTGTGGGCTTCCTGGAGACAATCTCACCTGGCTACTCCATCCACACCTACCTGTGGAGGAGGC AGTGA Codon-optimised SEQ ID NO: 7 - GBA Codon-optimised nucleotide GBA sequence nucleotide from sequence FLF-PL30, from with FLF-PL30, signal with signal peptide portion

ATGGAGTTTTCAAGTCCTTCCAGAGAGGAATGTCCCAAGCCTTTGAGTAGGGTAAGCATCATGGCT ATGGAGTTTTCAAGTCCTTCCAGAGAGGAATGTCCCAAGCCTTTGAGTAGGGTAAGCATCATGGCT GCAGCCTCACAGGATTGCTTCTACTTCAGGCAGTGTCGTGGGCATCAGGTGCCAGGCCCTGCAT GGCAGCCTCACAGGATTGCTTCTACTTCAGGCAGTGTCGTGGGCATCAGGTGCCAGGCCCTGCATC CCTAAGAGCTTTGGCTACAGCTCTGTGGTGTGTGTGTGCAATGCCACATACTGTGACTCCTTTGACC CCTAAGAGCTTTGGCTACAGCTCTGTGGTGTGTGTGTGCAATGCCACATACTGTGACTCCTTTGACC CCCCACCTTTCCTGCCCTGGGCACaTTctccAGaTATGAGAGCACAAGATCTGGGAGAAGGATGG, CCCCCACCTTTCCTGCCCTGGGCACaTTctccAGaTATGAGAGCACAAGATCTGGGAGAAGGATGGA GCTGAGCATGGGGCCCATCCAGGCTAATCACACTGGCACAGGCCTGCTGCTGACCCTGCAGCCTGA GCTGAGCATGGGGCCCATCCAGGCTAATCACACTGGCACAGGCCTGCTGCTGACCCTGCAGCCTGA ACAGAAGTTTCAGAAaGTGAAGGGATTTGGAGGGGCCATGACAGATGCTGCTGCTCTGAATATCC ACAGAAGTTTCAGAAaGTGAAGGGATTTGGAGGGGCCATGACAGATGCTGCTGCTCTGAATATCCT GGCCCTGTCACCCCCTGCCCAGAATCTGCTGCTGAAGAGCTACTTTTCAGAAGAAGGAATTGGATAT AATATCATCAGAGTGCCCATGGCCAGCTGTGACTTTTCCATCAGAACCTACACCTATGCAGACACO AATATCATCAGAGTGCCCATGGCCAGCTGTGACTTTTCCATCAGAACCTACACCTATGCAGACACCC CTGATGATTTTCAGCTGCACAATTTTAGCCTGCCTGAGGAAGATACCAAGCTGAAGATACCCCTGAT TCACAGGGCCCTGCAGCTGGCCCAGAGGCCTGTTTCACTGCTGGCCAGCCCCTGGACATCACCO TCACAGGGCCCTGCAGCTGGCCCAGAGGCCTGTTTCACTGCTGGCCAGCCCCTGGACATCACCCAC CTGGCTGAAGACCAATGGAGCTGTGAATGGGAAGGGGTCACTGAAGGGACAGCCTGGAGACATCTA CTGGCTGAAGACCAATGGAGCTGTGAATGGGAAGGGGTCACTGAAGGGACAGCCTGGAGACATCTA CCACCAGACCTGGGCCAGATACTTTGTGAAGTTTCTGGATGCCTATGCTGAGCACAAGCTGCAGTT CCACCAGACCTGGGCCAGATACTTTGTGAAGTTTCTGGATGCCTATGCTGAGCACAAGCTGCAGTTT TGGGCAGTGACAGCTGAAAATGAGCCTTCAGCTGGGCTGCTGTCAGGATACCCCTTTCAGTGCCTG ICTTTACCCCTGAACATCAGAGGGACTTTATTGCCAGGGACCTGGGCCCTACCCTGGCCAAT/ GGCTTTACCCCTGAACATCAGAGGGACTTTATTGCCAGGGACCTGGGCCCTACCCTGGCCAATAGC ACCCAcCAtAATGTgAGgttgCTGATGCTGGATGACCAGAGGCTGCTGCTGCCCCACTGGGCAAAGGT ACCCAcCAtAATGTgAGgttgCTGATGCTGGATGACCAGAGGCTGCTGCTGCCCCACTGGGCAAAGGT GGTGCTGACAGACCCTGAAGCAGCTAAaTATGTTCATGGCATTGCTGTGCATTGGTACCTGGACT GGTGCTGACAGACCCTGAAGCAGCTAAaTATGTTCATGGCATTGCTGTGCATTGGTACCTGGACTTT CTGGCTCCTGCCAAGGCCACCCTGGGGGAGACACACAGGCTGTTTCCCAATACCATGCTGTTTGCC CTGGCTCCTGCCAAGGCCACCCTGGGGGAGACACACAGGCTGTTTCCCAATACCATGCTGTTTGCO TCtGAGGCCTGTGTGGGCTCCAAGTTTTGGGAGCAGTCTGTGAGGCTGGGCTCCTGGGATAGAGO TCtGAGGCCTGTGTGGGCTCCAAGTTTTGGGAGCAGTCTGTGAGGCTGGGCTCCTGGGATAGAGG GATGCAGTACAGCCACAGCATCATCACCAATCTGCTGTACCATGTGGTGGGCTGGACTGACTGGA/ GATGCAGTACAGCCACAGCATCATCACCAATCTGCTGTACCATGTGGTGGGCTGGACTGACTGGAA CTGGCCCTGAATCCTGAAGGAGGACCtAAcTGGGTcAGgAATTTTGTGGACAGCCCCATCATTG) ACATCACCAAGGACACCTTTTACAAGCAGCCCATGTTTTACCACCTGGGCCACTTTAGCAAGTTT. GACATCACCAAGGACACCTTTTACAAGCAGCCCATGTTTTACCACCTGGGCCACTTTAGCAAGTTTA CCTGAGGGCTCCCAGAGAGTGGGGCTGGTTGCCAGCCAGAAGAATGACCTGGATGCAGTGGCA TTCCTGAGGGCTCCCAGAGAGTGGGGCTGGTTGCCAGCCAGAAGAATGACCTGGATGCAGTGGCAC TGATGCATCCTGATGGCTCAGCTGTTGTGGTGGTGCTGAATAGATCCAGCAAGGATGTGCCTCTGA TGATGCATCCTGATGGCTCAGCTGTTGTGGTGGTGCTGAATAGATCCAGCAAGGATGTGCCTCTGA CCATCAAGGATCCTGCTGTGGGCTTTCTGGAGACAATCTCACCTGGCTACTCCATTCACACCTACO CCATCAAGGATCCTGCTGTGGGCTTTCTGGAGACAATCTCACCTGGCTACTCCATTCACACCTACCT GTGGAGAAGGCAGTGA Codon-optimised SEQ ID NO: 8 - GBA Codon-optimised nucleotide GBA sequence nucleotide from sequence FLF-PL36, from with FLF-PL36, signal with signal peptide portion

GAGTTTTCAAGTCCTTCCAGAGAGGAATGTCCCAAGCCTTTGAGTAGGGTAAGCATCATO ATGGAGTTTTCAAGTCCTTCCAGAGAGGAATGTCCCAAGCCTTTGAGTAGGGTAAGCATCATGGCT GGCAGCCTCACAGGATTGCTTCTACTTCAGGCAGTGTCGTGGGCATCAGGTGCCAGGCCTTGCAT GGCAGCCTCACAGGATTGCTTCTACTTCAGGCAGTGTCGTGGGCATCAGGTGCCAGGCCTTGCATC CCAAAGTCTTTCGGCTACAGCTCCGTGGTGTGCGTGTGCAACGCCACCTATTGTGACTCCTTCGATO CCAAAGTCTTTCGGCTACAGCTCCGTGGTGTGCGTGTGCAACGCCACCTATTGTGACTCCTTCGATC CCCCTACCTTTCCCGCCCTGGGCACATTTTCTAGATACGAGTCTACACGCAGCGGCCGGAGAATGGA CCCCTACCTTTCCCGCCCTGGGCACATTTTCTAGATACGAGTCTACACGCAGCGGCCGGAGAATGGA GCTGAGCATGGGCCCTATCCAGGCCAATCACACAGGAACAGGCCTGCTGCTGACCCTGCAGCCAGA GCAGAAGTTCCAGAAGGTGAAGGGCTTTGGCGGAGCCATGACAGATGCAGCCGCCCTGAACATCO GCAGAAGTTCCAGAAGGTGAAGGGCTTTGGCGGAGCCATGACAGATGCAGCCGCCCTGAACATCCT GGCCCTGTCCCCACCCGCCCAGAATCTGCTGCTGAAGTCCTACTTCTCTGAGGAGGGCATCGGCTA TAACATCATCCGGGTGCCCATGGCCAGCTGCGACTTTTCCATCAGAACCTACACATATGCCGATAG CCTGACGATTTCCAGCTGCACAATTTTTCCCTGCCAGAGGAGGATACAAAGCTGAAGATCCCCCTO CCTGACGATTTCCAGCTGCACAATTTTTCCCTGCCAGAGGAGGATACAAAGCTGAAGATCCCCCTGA TCACCGGGCCCTGCAGCTGGCACAGCGGCCCGTGAGCCTGCTGGCCAGCCCCTGGACCTCCCCTA TTCACCGGGCCCTGCAGCTGGCACAGCGGCCCGTGAGCCTGCTGGCCAGCCCCTGGACCTCCCCTA CATGGCTGAAGACCAACGGCGCCGTGAATGGCAAGGGCTCTCTGAAGGGACAGCCTGGCGACAT CATGGCTGAAGACCAACGGCGCCGTGAATGGCAAGGGCTCTCTGAAGGGACAGCCTGGCGACATCT ACCACCAGACATGGGCCAGATATTTCGTGAAGTTTCTGGATGCCTACGCCGAGCACAAGCTGCAGTT ACCACCAGACATGGGCCAGATATTTCGTGAAGTTTCTGGATGCCTACGCCGAGCACAAGCTGCAGTT CTGGGCCGTGACAGCAGAGAATGAGCCTTCTGCCGGCCTGCTGAGCGGCTATCCCTTCCAGTGCCT CTGGGCCGTGACAGCAGAGAATGAGCCTTCTGCCGGCCTGCTGAGCGGCTATCCCTTCCAGTGCCT GGGCTLTACACCTGAGCACCAGCGGGACTTTATCGCCAGAGATCTGGGCCCAACCCTGGCCAACT GGGCTTTACACCTGAGCACCAGCGGGACTTTATCGCCAGAGATCTGGGCCCAACCCTGGCCAACTC

WO 2020/161483 wo PCT/GB2020/050251 13/22

CACACACCACAATGTGAGGCTGCTGATGCTGGACGATCAGCGCCTGCTGCTGCCTCACTGGGCCAA CACACACCACAATGTGAGGCTGCTGATGCTGGACGATCAGCGCCTGCTGCTGCCTCACTGGGCCAA GGTGGTGCTGACCGACCCAGAGGCCGCCAAGTACGTGCACGGCATCGCCGTGCACTGGTATCTGGA GGTGGTGCTGACCGACCCAGAGGCCGCCAAGTACGTGCACGGCATCGCCGTGCACTGGTATCTGGA MTTCCTGGCACCTGCAAAGGCCACCCTGGGAGAGACACACCGGCTGTTCCCTAACACCATGCTGTT7 GCCAGCGAGGCCTGCGTGGGCTCCAAGTTTTGGGAGCAGTCCGTGAGGCTGGGATCTTGGGACAC GCCAGCGAGGCCTGCGTGGGCTCCAAGTTTTGGGAGCAGTCCGTGAGGCTGGGATCTTGGGACAG AGGCATGCAGTACTCCCACTCTATCATCACCAATCTGCTGTATCACGTGGTGGGCTGGACAGACTGG AGGCATGCAGTACTCCCACTCTATCATCACCAATCTGCTGTATCACGTGGTGGGCTGGACAGACTGG AACCTGGCCCTGAATCCAGAGGGCGGCCCCAACTGGGTGAGAAATTTCGTGGATAGCCCCATCAT AACCTGGCCCTGAATCCAGAGGGCGGCCCCAACTGGGTGAGAAATTTCGTGGATAGCCCCATCATC TGGACATCACCAAGGATACATTCTACAAGCAGCCAATGTTTTATCACCTGGGCCACTTCTCTAAG GTGGACATCACCAAGGATACATTCTACAAGCAGCCAATGTTTTATCACCTGGGCCACTTCTCTAAGT TATCCCTGAGGGCAGCCAGAGGGTGGGCCTGGTGGCCAGCCAGAAGAACGACCTGGATGCCGTG TTATCCCTGAGGGCAGCCAGAGGGTGGGCCTGGTGGCCAGCCAGAAGAACGACCTGGATGCCGTG GCCCTGATGCACCCTGATGGCTCCGCCGTGGTGGTGGTGCTGAATCGCTCTAGCAAGGACGTGC< GCCCTGATGCACCCTGATGGCTCCGCCGTGGTGGTGGTGCTGAATCGCTCTAGCAAGGACGTGCCT TGACCATCAAGGATCCAGCCGTGGGATTTCTGGAGACTATTTCACCTGGCTATTCAATTCATACCT CTGACCATCAAGGATCCAGCCGTGGGATTTCTGGAGACTATTTCACCTGGCTATTCAATTCATACCT ACCTGTGGAGGAGGCAGTGA SEQ ID NO: 9 - Wild type human GBA nucleotide sequence with signal peptide (from GenBank NM_000157.3)

ATGGAGTTTTCAAGTCCTTCCAGAGAGGAATGTCCCAAGCCTTTGAGTAGGGTAAGCATCATGGCT ATGGAGTTTTCAAGTCCTTCCAGAGAGGAATGTCCCAAGCCTTTGAGTAGGGTAAGCATCATGGCT GGCAGCCTCACAGGATTGCTTCTACTTCAGGCAGTGTCGTGGGCATCAGGTGCCCGCCCCTGCATO GGCAGCCTCACAGGATTGCTTCTACTTCAGGCAGTGTCGTGGGCATCAGGTGCCCGCCCCTGCATO CCTAAAAGCTTCGGCTACAGCTCGGTGGTGTGTGTCTGCAATGCCACATACTGTGACTCCTTTGACO CCTAAAAGCTTCGGCTACAGCTCGGTGGTGTGTGTCTGCAATGCCACATACTGTGACTCCTTTGACC CCCCGACCTTTCCTGCCCTTGGTACCTTCAGCCGCTATGAGAGTACACGCAGTGGGCGACGGATG AGCTGAGTATGGGGCCCATCCAGGCTAATCACACGGGCACAGGCCTGCTACTGACCCTGCAGCCA AGCTGAGTATGGGGCCCATCCAGGCTAATCACACGGGCACAGGCCTGCTACTGACCCTGCAGCCAG AACAGAAGTTCCAGAAAGTGAAGGGATTTGGAGGGGCCATGACAGATGCTGCTGCTCTCAACATCO AACAGAAGTTCCAGAAAGTGAAGGGATTTGGAGGGGCCATGACAGATGCTGCTGCTCTCAACATCO TTGCCCTGTCACCCCCTGCCCAAAATTTGCTACTTAAATCGTACTTCTCTGAAGAAGGAATCGGATA TTGCCCTGTCACCCCCTGCCCAAAATTTGCTACTTAAATCGTACTTCTCTGAAGAAGGAATCGGATA TAACATCATCCGGGTACCCATGGCCAGCTGTGACTTCTCCATCCGCACCTACACCTATGCAGACAG TAACATCATCCGGGTACCCATGGCCAGCTGTGACTTCTCCATCCGCACCTACACCTATGCAGACACO ECTGATGATTTCCAGTTGCACAACTTCAGCCTCCCAGAGGAAGATACCAAGCTCAAGATACCCCTGA CCTGATGATTTCCAGTTGCACAACTTCAGCCTCCCAGAGGAAGATACCAAGCTCAAGATACCCCTGA TCACCGAGCCCTGCAGTTGGCCCAGCGTCCCGTTTCACTCCTTGCCAGCCCCTGGACATCACCCA0 TTCACCGAGCCCTGCAGTTGGCCCAGCGTCCCGTTTCACTCCTTGCCAGCCCCTGGACATCACCCAG TTGGCTCAAGACCAATGGAGCGGTGAATGGGAAGGGGTCACTCAAGGGACAGCCCGGAGACATCTA TTGGCTCAAGACCAATGGAGCGGTGAATGGGAAGGGGTCACTCAAGGGACAGCCCGGAGACATCTA CCACCAGACCTGGGCCAGATACTTTGTGAAGTTCCTGGATGCCTATGCTGAGCACAAGTTACAGTT CCACCAGACCTGGGCCAGATACTTTGTGAAGTTCCTGGATGCCTATGCTGAGCACAAGTTACAGTTC TGGGCAGTGACAGCTGAAAATGAGCCTTCTGCTGGGCTGTTGAGTGGATACCCCTTCCAGTGCCT TGGGCAGTGACAGCTGAAAATGAGCCTTCTGCTGGGCTGTTGAGTGGATACCCCTTCCAGTGCCTG GGCTTCACCCCTGAACATCAGCGAGACTTCATTGCCCGTGACCTAGGTCCTACCCTCGCCAACAGT GGCTTCACCCCTGAACATCAGCGAGACTTCATTGCCCGTGACCTAGGTCCTACCCTCGCCAACAGTA TCACCACAATGTCCGCCTACTCATGCTGGATGACCAACGCTTGCTGCTGCCCCACTGGGCAAAGO CTCACCACAATGTCCGCCTACTCATGCTGGATGACCAACGCTTGCTGCTGCCCCACTGGGCAAAGGT GGTACTGACAGACCCAGAAGCAGCTAAATATGTTCATGGCATTGCTGTACATTGGTACCTGGACTTI GGTACTGACAGACCCAGAAGCAGCTAAATATGTTCATGGCATTGCTGTACATTGGTACCTGGACTTT

90 CTGGCTCCAGCCAAAGCCACCCTAGGGGAGACACACCGCCTGTTCCCCAACACCATGCTCTTTGCC CTGGCTCCAGCCAAAGCCACCCTAGGGGAGACACACCGCCTGTTCCCCAACACCATGCTCTTTGCCT PAGAGGCCTGTGTGGGCTCCAAGTTCTGGGAGCAGAGTGTGCGGCTAGGCTCCTGGGATCGAGGe CAGAGGCCTGTGTGGGCTCCAAGTTCTGGGAGCAGAGTGTGCGGCTAGGCTCCTGGGATCGAGGG ATGCAGTACAGCCACAGCATCATCACGAACCTCCTGTACCATGTGGTCGGCTGGACCGACTGGAACO ATGCAGTACAGCCACAGCATCATCACGAACCTCCTGTACCATGTGGTCGGCTGGACCGACTGGAACC TTGCCCTGAACCCCGAAGGAGGACCCAATTGGGTGCGTAACTTTGTCGACAGTCCCATCATTGTAGA CATCACCAAGGACACGTTLTACAAACAGCCCATGTTCTACCACCTTGGCCACTTCAGCAAGTTCATTO CATCACCAAGGACACGTTTTACAAACAGCCCATGTTCTACCACCTTGGCCACTTCAGCAAGTTCATTC CTGAGGGCTCCCAGAGAGTGGGGCTGGTTGCCAGTCAGAAGAACGACCTGGACGCAGTGGCACT CTGAGGGCTCCCAGAGAGTGGGGCTGGTTGCCAGTCAGAAGAACGACCTGGACGCAGTGGCACTG ATGCATCCCGATGGCTCTGCTGTTGTGGTCGTGCTAAACCGCTCCTCTAAGGATGTGCCTCTTACCA ATGCATCCCGATGGCTCTGCTGTTGTGGTCGTGCTAAACCGCTCCTCTAAGGATGTGCCTCTTACCA CAAGGATCCTGCTGTGGGCTTCCTGGAGACAATCTCACCTGGCTACTCCATTCACACCTACCTGTG TCAAGGATCCTGCTGTGGGCTTCCTGGAGACAATCTCACCTGGCTACTCCATTCACACCTACCTGTG GCGTCGCCAGTGA

SEQ ID NO: 10 - LSP-S LSP-S transcription transcription regulatory regulatory element element

CCCTAAAATGGGCAAACATTGCAAGCAGCAAACAGCAAACACACAGCCCTCCCTGCCTGCTGACCT CCCTAAAATGGGCAAACATTGCAAGCAGCAAACAGCAAACACACAGCCCTCCCTGCCTGCTGACCTT GGAGCTGGGGCAGAGGTCAGACACCTCTCTGGGCCCATGCCACCTCCAACTGGACACAGGACGC GGAGCTGGGGCAGAGGTCAGACACCTCTCTGGGCCCATGCCACCTCCAACTGGACACAGGACGCTG TGGTTTCTGAGCCAGGGGGCGACTCAGATCCCAGCCAGTGGACTTAGCCCCTGTTTGCTCCTCCGA TGGTTTCTGAGCCAGGGGGCGACTCAGATCCCAGCCAGTGGACTTAGCCCCTGTTTGCTCCTCCGA AACTGGGGTGACCTTGGTTAATATTCACCAGCAGCCTCCCCCGTTGCCCCTCTGGATCCACTGCT TAACTGGGGTGACCTTGGTTAATATTCACCAGCAGCCTCCCCCGTTGCCCCTCTGGATCCACTGCTT AAATACGGACGAGGACAGGGCCCTGTCTCCTCAGCTTCAGGCACCACCACTGACCTGGGACAGTGA AAATACGGACGAGGACAGGGCCCTGTCTCCTCAGCTTCAGGCACCACCACTGACCTGGGACAGTGA AT SEQ ID NO: 11 - HCR HCR enhancer enhancer portion portion ofof LSP-S LSP-S wo 2020/161483 WO PCT/GB2020/050251 PCT/GB2020/050251 14/22

CCCTAAAATGGGCAAACATTGCAAGCAGCAAACAGCAAACACACAGCCCTCCCTGCCTGCTGACCT CCCTAAAATGGGCAAACATTGCAAGCAGCAAACAGCAAACACACAGCCCTCCCTGCCTGCTGACCTI GGAGCTGGGGCAGAGGTCAGACACCTCTCTGGGCCCATGCCACCTCCAAC GGAGCTGGGGCAGAGGTCAGACACCTCTCTGGGCCCATGCCACCTCCAAC

SEQ ID NO: 12 - A1AT A1AT promoter promoter portion portion ofof LSP-S LSP-S

GGGCGACTCAGATCCCAGCCAGTGGACTTAGCCCCTGTTTGCTCCTCCGATAACTGGGGTGACCT GGGCGACTCAGATCCCAGCCAGTGGACTTAGCCCCTGTTTGCTCCTCCGATAACTGGGGTGACCTT GGTTAATATTCACCAGCAGCCTCCCCCGTTGCCCCTCTGGATCCACTGCTTAAATACGGACGAGGA0 GGTTAATATTCACCAGCAGCCTCCCCCGTTGCCCCTCTGGATCCACTGCTTAAATACGGACGAGGAC AGGGCCCTGTCTCCTCAGCTTCAGGCACCACCACTGACCTGGGACAGTGAAT AGGGCCCTGTCTCCTCAGCTTCAGGCACCACCACTGACCTGGGACAGTGAAT

SEQ ID SEQ ID NO: NO:1313- CAG promoter CAG promoter

GACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATG GACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATO GAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCAT GAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCAT GACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGT GGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCT GGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCT ATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTT ATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTC CTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCT6 CTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTG

GCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGG TGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGG GGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTT GGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTT TCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCC TCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCG

SEQ ID NO: 14 - LSP-L LSP-L transcription transcription regulatory regulatory element element

AGGCTCAGAGGCACACAGGAGTTTCTGGGCTCACCCTGCCCCCTTCCAACCCCTCAGTTCCCATCCT CCAGCAGCTGTTTGTGTGCTGCCTCTGAAGTCCACACTGAACAAACTTCAGCCTACTCATGTCCO CCAGCAGCTGTTTGTGTGCTGCCTCTGAAGTCCACACTGAACAAACTTCAGCCTACTCATGTCCCTA AAATGGGCAAACATTGCAAGCAGCAAACAGCAAACACACAGCCCTCCCTGCCTGCTGACCTTGGAGO AAATGGGCAAACATTGCAAGCAGCAAACAGCAAACACACAGCCCTCCCTGCCTGCTGACCTTGGAGG GGGGCAGAGGTCAGAGACCTCTCTGGGCCCATGCCACCTCCAACATCCACTCGACCCCTTGGAAT TGGGGCAGAGGTCAGAGACCTCTCTGGGCCCATGCCACCTCCAACATCCACTCGACCCCTTGGAATT TCGGTGGAGAGGAGCAGAGGTTGTCCTGGCGTGGTTTAGGTAGTGTGAGAGGGGTACCCGGGGAT CTTGCTACCAGTGGAACAGCCACTAAGGATTCTGCAGTGAGAGCAGAGGGCCAGCTAAGTGGTACT CTTGCTACCAGTGGAACAGCCACTAAGGATTCTGCAGTGAGAGCAGAGGGCCAGCTAAGTGGTACT CTCCCAGAGACTGTCTGACTCACGCCACCCCCTCCACCTTGGACACAGGACGCTGTGGTTTCTGA0 CTCCCAGAGACTGTCTGACTCACGCCACCCCCTCCACCTTGGACACAGGACGCTGTGGTTTCTGAGC CAGGTACAATGACTCCTTTCGGTAAGTGCAGTGGAAGCTGTACACTGCCCAGGCAAAGCGTCCGGG CAGGTACAATGACTCCTTTCGGTAAGTGCAGTGGAAGCTGTACACTGCCCAGGCAAAGCGTCCGGG CAGCGTAGGCGGGCGACTCAGATCCCAGCCAGTGGACTTAGCCCCTGTTTGCTCCTCCGATAACT CAGCGTAGGCGGGCGACTCAGATCCCAGCCAGTGGACTTAGCCCCTGTTTGCTCCTCCGATAACTG GGGTGACCTTGGTTAATATTCACCAGCAGCCTCCCCCGTTGCCCCTCTGGATCCACTGCTTAAATAG GGGTGACCTTGGTTAATATTCACCAGCAGCCTCCCCCGTTGCCCCTCTGGATCCACTGCTTAAATAC GGACGAGGACAGGGCCCTGTCTCCTCAGCTTCAGGCACCACCACTGACCTGGGACAGTGAATGATC CCCCTGATCTGCGGCC A1AT SEQ ID NO: 15 - promoter A1AT portion promoter ofof portion LSP-L LSP-L

GGATCTTGCTACCAGTGGAACAGCCACTAAGGATTCTGCAGTGAGAGCAGAGGGCCAGCTAAGT GGATCTTGCTACCAGTGGAACAGCCACTAAGGATTCTGCAGTGAGAGCAGAGGGCCAGCTAAGTGG TACTCTCCCAGAGACTGTCTGACTCACGCCACCCCCTCCACCTTGGACACAGGACGCTGTGGTTTCT TACTCTCCCAGAGACTGTCTGACTCACGCCACCCCCTCCACCTTGGACACAGGACGCTGTGGTTTCT

11 GAGCCAGGTACAATGACTCCTTTCGGTAAGTGCAGTGGAAGCTGTACACTGCCCAGGCAAAGCGT0 GAGCCAGGTACAATGACTCCTTTCGGTAAGTGCAGTGGAAGCTGTACACTGCCCAGGCAAAGCGTC CGGGCAGCGTAGGCGGGCGACTCAGATCCCAGCCAGTGGACTTAGCCCCTGTTTGCTCCTCCGATA CGGGCAGCGTAGGCGGGCGACTCAGATCCCAGCCAGTGGACTTAGCCCCTGTTTGCTCCTCCGATA ACTGGGGTGACCTTGGTTAATATTCACCAGCAGCCTCCCCCGTTGCCCCTCTGGATCCACTGCTTA/ ACTGGGGTGACCTTGGTTAATATTCACCAGCAGCCTCCCCCGTTGCCCCTCTGGATCCACTGCTTAA ATACGGACGAGGACAGGGCCCTGTCTCCTCAGCTTCAGGCACCACCACTGACCTGGGACAGTGAAT ATACGGACGAGGACAGGGCCCTGTCTCCTCAGCTTCAGGCACCACCACTGACCTGGGACAGTGAAT GATCCCCCTGATCTGCGGCC

SEQ ID NO: 16 - HCR HCR enhancer enhancer portion portion ofof LSP-L LSP-L

AGGCTCAGAGGCACACAGGAGTTTCTGGGCTCACCCTGCCCCCTTCCAACCCCTCAGTTCCCATCCT AGGCTCAGAGGCACACAGGAGTTTCTGGGCTCACCCTGCCCCCTTCCAACCCCTCAGTTCCCATCCT CCAGCAGCTGTTTGTGTGCTGCCTCTGAAGTCCACACTGAACAAACTTCAGCCTACTCATGTCCCT CCAGCAGCTGTTTGTGTGCTGCCTCTGAAGTCCACACTGAACAAACTTCAGCCTACTCATGTCCCTA AATGGGCAAACATTGCAAGCAGCAAACAGCAAACACACAGCCCTCCCTGCCTGCTGACCTTGGAG AAATGGGCAAACATTGCAAGCAGCAAACAGCAAACACACAGCCCTCCCTGCCTGCTGACCTTGGAGG GGGGCAGAGGTCAGAGACCTCTCTGGGCCCATGCCACCTCCAACATCCACTCGACCCCTTGGAAT TGGGGCAGAGGTCAGAGACCTCTCTGGGCCCATGCCACCTCCAACATCCACTCGACCCCTTGGAATT TCGGTGGAGAGGAGCAGAGGTTGTCCTGGCGTGGTTTAGGTAGTGTGAGAGGG TCGGTGGAGAGGAGCAGAGGTTGTCCTGGCGTGGTTTAGGTAGTGTGAGAGGG wo 2020/161483 WO PCT/GB2020/050251 15/22

Wild SEQ ID NO: 17 - type Wild GBA type nucleotide GBA sequence nucleotide corresponding sequence toto corresponding signal peptide signal peptide

ATGGAGTTTTCAAGTCCTTCCAGAGAGGAATGTCCCAAGCCTTTGAGTAGGGTAAGCATCATGGCT ATGGAGTTTTCAAGTCCTTCCAGAGAGGAATGTCCCAAGCCTTTGAGTAGGGTAAGCATCATGGO GGCAGCCTCACAGGATTGCTTCTACTTCAGGCAGTGTCGTGGGCATCAGGT GGCAGCCTCACAGGATTGCTTCTACTTCAGGCAGTGTCGTGGGCATCAGGT Wild SEQ ID NO: 18 - type Wild GCase type polypeptide GCase sequence polypeptide ofof sequence signal peptide signal peptide

MEFSSPSREECPKPLSRVSIMAGSLTGLLLLQAVSWASG MEFSSPSREECPKPLSRVSIMAGSLTGLLLLQAVSWASG

SEQ ID NO: 19 - Polypeptide sequence of liver-tropic capsid

ADGYLPDWLEDNLSEGIREWWALKPGVPQPKANQQHQDNRRGLVLPGYKYLGPGNGLDKGEP) MAADGYLPDWLEDNLSEGIREWWALKPGVPQPKANQQHQDNRRGLVLPGYKYLGPGNGLDKGEPVNEA DAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRILEPLGLVEEAAKT APGKKRPVDQSPQEPDSSSGVGKSGKQPARKRLNFGQTGDSESVPDPOPLGEPPAAPTSLGSNTMASGGG APGKKRPVDQSPQEPDSSSGVGKSGKQPARKRLNFGQTGDSESVPDPQPLGEPPAAPTSLGSNTMASGGO APMADNNEGADGVGNSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYST APMADNNEGADGVGNSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYST PWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLSFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSE PWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLSFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSE YQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYTFED YQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYTFED VPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQGTTSGTTNQSRLLFSQAGPQSMSLQARNWLPGPCYRQQ VPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQGTTSGTTNQSRLLFSQAGPOSMSLQARNWLPGPCYRQQ RLSKTANDNNNSNFPWTAASKYHLNGRDSLVNPGPAMASHKDDEEKFFPMHGNLIFGKEGTTASNAELDI RLSKTANDNNNSNFPWTAASKYHLNGRDSLVNPGPAMASHKDDEEKFFPMHGNLIFGKEGTTASNAELDN /MITDEEEIRTTNPVATEQYGTVANNLQSSNTAPTTRTVNDQGALPGMVWQDRDVYLQGPIWAKIPHTD VMITDEEEIRTTNPVATEQYGTVANNLQSSNTAPTTRTVNDQGALPGMVWQDRDVYLQGPIWAKIPHTD GHFHPSPLMGGFGLKHPPPQIMIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNP GHFHPSPLMGGFGLKHPPPQIMIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNP EIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL EIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

SEQ ID NO: 20 - Polypeptide Polypeptide sequence sequence ofof liver-tropic liver-tropic capsid capsid

AAADGYLPDWLEDNLSEGIREWWALKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVN DAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEG DAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAK TAPGKKRPVDQSPQEPDSSSGVGKSGKQPARKRLNFGQTGDSESVPDPQPLGEPPAAPTSLGSNTMASGO TAPGKKRPVDQSPQEPDSSSGVGKSGKQPARKRLNFGOTGDSESVPDPQPLGEPPAAPTSLGSNTMASGG APMADNNEGADGVGNSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGY GAPMADNNEGADGVGNSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGY STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLSFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTD SEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYTF SEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYTF EDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQGTTSGTTNQSRLLFSQAGPQSMSLQARNWLPGPCYI EDVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQGTTSGTTNQSRLLFSQAGPQSMSLQARNWLPGPCYR QRLSKTANDNNNSNFPWTAASKYHLNGRDSLVNPGPAMASHKDDEEKFFPMHGNLIFGKEGTTASNAL QQRLSKTANDNNNSNFPWTAASKYHLNGRDSLVNPGPAMASHKDDEEKFFPMHGNLIFGKEGTTASNAEL DNVMITDEEEIRTTNPVATEQYGTVANNLQSSNTAPTTRTVNDQGALPGMVWQDRDVYLQGPIWAKIPHT DNVMITDEEEIRTTNPVATEQYGTVANNLQSSNTAPTTRTVNDQGALPGMVWQDRDVYLQGPIWAKIPHT GHFHPSPLMGGFGLKHPPPQIMIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWN DGHFHPSPLMGGFGLKHPPPQIMIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWN EIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL PEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL

SEQ ID NO: 21 - Polypeptide Polypeptide sequence sequence ofof CNS-tropic CNS-tropic capsid capsid

MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAA MAADGYLPDWLEDNLSEGIREWWALKPGAPQPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAA DAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEE DAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKT APGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGG APGKKRPVEQSPQEPDSSAGIGKSGAQPAKKRLNFGQTGDTESVPDPQPIGEPPAAPSGVGSLTMASGGG APVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFGY IPVADNNEGADGVGSSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISNSTSGGSSNDNAYFG) STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTD STPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTDNNGVKTIANNLTSTVQVFTD SDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYEFI SDYQLPYVLGSAHEGCLPPFPADVFMIPQYGYLTLNDGSQAVGRSSFYCLEYFPSOMLRTGNNFOFSYEFE NVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQORV NVPFHSSYAHSQSLDRLMNPLIDQYLYYLSKTINGSGQNQQTLKFSVAGPSNMAVQGRNYIPGPSYRQQRV STTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADK STTVTQNNNSEFAWPGASSWALNGRNSLMNPGPAMASHKEGEDRFFPLSGSLIFGKQGTGRDNVDADKV MITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTD MITNEEEIKTTNPVATESYGQVATNHQSAQAQAQTGWVQNQGILPGMVWQDRDVYLQGPIWAKIPHTDG NFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPE NFHPSPLMGGFGMKHPPPQILIKNTPVPADPPTAFNKDKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEI QYTSNYYKSNNVEFAVNTEGVYSEPRPIGTRYLTRNL

SEQ ID NO: 22 - Nucleotide Nucleotide sequence sequence ofof SV40 SV40 intron intron wo 2020/161483 WO PCT/GB2020/050251 16/22

GTAAATATAAAATTTTTAAGTGTATAATGTGTTAAACTACTGATTCTAATTGTTTCTCTCTTTTAG GTAAATATAAAATTTTTAAGTGTATAATGTGTTAAACTACTGATTCTAATTGTTTCTCTCTTTTAG

Nucleotide SEQ ID NO: 23 - sequence Nucleotide ofof sequence bovine growth bovine hormone growth poly hormone A A poly sequence sequence

TGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAG CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGG TGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCAT CTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGO TCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCA TGCTGGGGA SEQ ID NO: 24 - Polypeptide sequence of liver-tropic capsid

MAADGYLPDWLEDNLSEGIREWWALQPGAPKPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAA MAADGYLPDWLEDNLSEGIREWWALQPGAPKPKANQQHQDNARGLVLPGYKYLGPGNGLDKGEPVNAA DAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLVEEAAKT DAAALEHDKAYDQQLKAGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRLLEPLGLYEEAAKT APGKKRPVDQSPQEPDSSSGVGKSGKQPARKRLNFGQTGDSESVPDPQPLGEPPAAPTSLGSNTMASGGG APGKKRPVDQSPQEPDSSSGVGKSGKQPARKRLNFGQTGDSESVPDPQPLGEPPAAPTSLGSNTMASGGG PMADNNEGADGVGNSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYST APMADNNEGADGVGNSSGNWHCDSQWLGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYST WGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLSFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSE PWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKKLSFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSE YQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYTFED YQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFSYTFED VPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQGTTSGTTNQSRLLFSQAGPQSMSLQARNWLPGPCYRQQ RLSKTANDNNNSNFPWTAASKYHLNGRDSLVNPGPAMASHKDDEEKFFPMHGNLIFGKEGTTASNAELDN RLSKTANDNNNSNFPWTAASKYHLNGRDSLVNPGPAMASHKDDEEKFFPMHGNLIFGKEGTTASNAELDNI (MITDEEEIRTTNPVATEQYGTVANNLQSSNTAPTTRTVNDQGALPGMVWQDRDVYLQGPIWAKIPHTD VMITDEEEIRTTNPVATEQYGTVANNLQSSNTAPTTRTVNDQGALPGMVWQDRDVYLQGPIWAKIPHTD GHFHPSPLMGGFGLKHPPPQIMIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNP GHFHPSPLMGGFGLKHPPPQIMIKNTPVPANPPTTFSPAKFASFITQYSTGQVSVEIEWELQKENSKRWNP EIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRPL EIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRPL

SEQ SEQ ID ID NO: NO:2525- Polypeptide Polypeptide sequence sequenceof of wild typetype wild human GCaseGCase human

MEFSSPSREECPKPLSRVSIMAGSLTGLLLLQAVSWASGARPCIPKSFGYSSVVCVCNATYCDSFDPPT MEFSSPSREECPKPLSRVSIMAGSLTGLLLLQAVSWASGARPCIPKSFGYSSVVCVCNATYCDSFDPPTFPAL GTFSRYESTRSGRRMELSMGPIQANHTGTGLLLTLQPEQKFQKVKGFGGAMTDAAALNILALSPPAQNLLE GTFSRYESTRSGRRMELSMGPIQANHTGTGLLLTLQPEQKFQKVKGFGGAMTDAAALNILALSPPAQNLLL KSYFSEEGIGYNIIRVPMASCDFSIRTYTYADTPDDFQLHNFSLPEEDTKLKIPLIHRALQLAQRPVSLLASPW TSPTWLKTNGAVNGKGSLKGQPGDIYHQTWARYFVKFLDAYAEHKLQFWAVTAENEPSAGLLSGYPFQC TSPTWLKTNGAVNGKGSLKGQPGDIYHQTWARYFVKFLDAYAEHKLQFWAVTAENEPSAGLLSGYPFQC LGFTPEHQRDFIARDLGPTLANSTHHNVRLLMLDDQRLLLPHWAKVVLTDPEAAKYVHGIAVHWYLDFLA LGFTPEHQRDFIARDLGPTLANSTHHNVRLLMLDDQRLLLPHWAKVVLTDPEAAKYVHGIAVHWYLDFLA PAKATLGETHRLFPNTMLFASEACVGSKFWEQSVRLGSWDRGMQYSHSIITNLLYHVVGWTDWNLALNPE PAKATLGETHRLFPNTMLFASEACVGSKFWEQSVRLGSWDRGMQYSHSITNLLYHVVGWTDWNLALNPE GGPNWVRNFVDSPIIVDITKDTFYKQPMFYHLGHFSKFIPEGSQRVGLVASQKNDLDAVALMHPDGSAVVV LNRSSKDVPLTIKDPAVGFLETISPGYSIHTYLWRRQ VLNRSSKDVPLTIKDPAVGFLETISPGYSIHTYLWRRQ

FIGURE 10

A 6 6 (nmol/hr/ml) activity GCase 5 5

4

3 3

2

1 1

0 HUH7 HUH7 HEK293T HEK293TPANC-1 BxPC3 MCF-7 PANC-1 BxPC3 1643 MRC-9 MCF-7 1643 MRC-9697 697

B 10 cell / genome Viral 103 10³

102 10²

10¹ 10

10° HUH7 HUH7HEK293T HEK293TPANC-1 BxPC3 PANC-1 MCF71643 MCF7 BxPC3 1643 MRC9 MRC9 697

WO wo 2020/161483 PCT/GB2020/050251 18/22

FIGURE 11

A GCase activity (µmol/h/ml)

25 Best-fit values

YO Y0 18.27

20 Plateau -0.1194

K 0.1248 Half Life 5.553 5.553 15 Tau 8.012 Span 18.39 10

5

0 0 and

1 10 100 1000 10000 Minutes (log10) Minutes (log)

B

GCase activity (µmol/h/ml) 20

15 ERT (60 U/kg) FLF-PL64 (2x1012 FLF-PL64 12 vg/kg) (2x10¹² vg/kg) - - - 10

5 5

0 0 30 60 2000 4000 6000 8000 10000 Time post infusion (min)

PCT/GB2020/050251 19/22

FIGURE 12

Liver Spleen Bone

Naive

20 min

60 min

VPRIV 60U/kg 240 min

1440 min

FLF-PL64 5-weeks p.i. 2x1012 2x10¹² vg/kg

GCase (brown); Haematoxylin Haemataxylin (blue)

FIGURE 13

a 150 200 200 Liver WBCs

type wild from % type wild from % 150 100

100

50 50 50

0 0 Vehicle AAV-GBA ERT Vehicle AAV-GBA ERT

C 250 d 200 Spleen Bone marrow 200 type wild from % type wild from % 150

150 150 I 100 100 100 **** 50 50 50

8 0 0 Vehicle AAV-GBA ERT Vehicle AAV-GBA ERT

FIGURE 14

Vehicle the Wild-type

Vehicle Vehicle

Gba 9v/null Gbav/null AAV-GBA AAVEBA

gba 9V/null mice gbaV/null mice gba 9V/null mice gbaV/null mice vehicle) of (% cells storage of Number / **** vehicle) of (% cells Positive CD68 100 100

50 50

0 0 Vehicle VehicleAAV-GBA AAV-GBA ERT ERT Vehicle AAV-GBA AAV-GBA ERT

WO wo 2020/161483 PCT/GB2020/050251 PCT/GB2020/050251 22/22

FIGURE 15

Hexosylceramide 200 treated vehicle from % Vehicle 150 ERT II AAV-GBA 100

50

% 0 0 Liver Spleen Bone marrow

Hexosylsphingosine * * * 150

% from vehicle treated

Vehicle

100 ERT AAV-GBA T

50

% 0 Liver Spleen Bone marrow